Intercellular labeling of ligand-receptor interactions

ABSTRACT

An sortase-mediated intercellular labeling method allowing for tracking ligand-receptor interaction both in vitro and in vivo; and uses thereof for tracking molecule interactions both in vitro and in vivo, identifying modulators of ligand-receptor interaction, identifying potential binding partners of a protein of interest, identifying B cells expressing high affinity B cell receptors to antigens, and identifying the antigen to which a T cell of interest binds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority under 35 U.S.C.§ 120 to U.S. application Ser. No. 14/875,140, filed Oct. 5, 2015, whichclaims the benefit of the filing date of U.S. Provisional ApplicationNo. 62/059,452, filed Oct. 3, 2014, the entire contents of which areincorporated by reference herein.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.1DP5OD01214601, awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

One of the key characteristics of the immune system is the ability toreact to pathogens by generating antibodies. An effective immuneresponse requires the production of antibodies that bind antigens withhigh affinity and specificity. Exposure to antigen triggers thegeneration and clonal selection of B cells carrying novel mutant Igsequences with improved antigen affinity, in a phenomenon known asaffinity maturation. Affinity maturation is the result of thecombination of two processes: somatic hypermutation and affinity-basedselection, both of which occur in anatomic structures referred to asgerminal centers. Recent studies suggest that the antigen-dependentinteraction between B cells at germinal centers and follicular T helper(Tfh) cells, which are the limiting factor in affinity-based selectionof B cells that express high affinity antibodies. According to theproposed model, B cells that exhibit high affinity Ig molecules at theplasma membrane will capture and process more antigen for presentationon major histocompatibility complex (MHC) class II molecules. A limitednumber of Tfh cells then selects those B cells with the highestpeptide-MHC density and directs their return to the dark zones ingerminal centers, where the selected B cells undergo rapid division. Bycontrast, B cells that fail to interact with Tfh cells undergoapoptosis.

Despite the crucial role of the interaction between Tfh cells and Bcells in affinity maturation, little is known about how theseinteractions lead to selection of some cells and elimination of othersin an in vivo setting. This gap is due largely to the fact that there isno effective way to determine the extent to which two cells haveinteracted within a living animal. Interactions between differentligand-receptor pairs expressed by various subsets of immune cells arekey events in the immune response, but the tracking of theseinteractions in the context of a living animal has never been achieved.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides an intercellular labelingmethod, which comprises (i) providing a first cell expressing a firstpolypeptide on its surface, the first polypeptide comprising a sortaseacceptor peptide (e.g., GGG), which is located at the N-terminus of thefirst polypeptide; (ii) providing a second cell expressing a secondpolypeptide on its surface, the second polypeptide comprising a sortase(e.g., a sortase A, a sortase B, a sortase C, or a sortase D) or anactive fragment thereof; and (iii) contacting the first cell with thesecond cell in the presence of a peptide comprising a sortaserecognition sequence (e.g., LPTXG (SEQ ID NO: 4) for sortase A), whereinthe peptide is associated with a detectable label, e.g., biotin or afluorescent dye. In some examples, the first polypeptide may be a fusionpolypeptide that comprises the sortase acceptor peptide and one memberof a receptor-ligand pair. Alternatively or in addition, the secondpolypeptide may also be a fusion polypeptide that comprises the sortaseor the active fragment thereof and the other member of thereceptor-ligand pair.

Exemplary receptor-ligand pairs include, but are not limited to, CD40and CD40L, CD80 and CD28, CD80 and CTLA4, CD86 and CD28, CD86 and CTLA4,PD-1 and PD-L1, PD-1 and PD-L2, or ICOS and ICOSL. Upon interactionbetween the first cell and the second cell (e.g., via thereceptor-ligand pair), the sortase, or the active fragment thereof linksthe peptide to the first polypeptide, thereby labeling the first cellexpressing the first polypeptide. The first polypeptide, the secondpolypeptide, or both may further comprise a protein tag, which mayfacilitate purification and isolation of the polypeptide comprising suchor cells expressing the polypeptide.

In some embodiments, the first cell, the second cell, or both are immunecells, for example, T cells, B cells, dendritic cells, macrophages, ornatural killer cells. In one example, the first cell is a T cell, andthe second cell is a B cell, or vice versa.

In some embodiments, the sortase used in the intercellular labelingmethod is a mutant sortase (e.g., a mutant sortase A) that exhibitsimproved catalytic activity as compared to its wild-type counterpart. Insome examples, the mutant sortase A (SrtA) comprises one or moremutations of P94R or P94S, S102C, A104H, E105D, K138P, K152I, D160K orD160N, K162H, T164N, D165A, K173E, I182V, K190E, and K196S or K196T. Inone example, the mutant SrtA includes mutations P94S, D160N, and K196T.

In any of the intercellular labeling methods described herein, thesortase recognition sequence is LPXTG (SEQ ID NO: 1), in which X is anyamino acid residue. For example, the sortase recognition sequence isLPETG (SEQ ID NO: 2), which may be co-used with any of the sortase Aenzymes, including both wild-type SrtAs and SrtA mutants disclosedherein or known in the art. Alternatively or in addition, the sortaseacceptor peptide may be an oligoglycine, e.g., consisting of 1-5 glycineresidues.

In some examples, the intercellular labeling method as described hereininvolves a first polypeptide comprising CD40 and oligoglycine GGGGG (SEQID NO: 3), which is located at the N-terminus of the first polypeptide,and a second polypeptide comprising CD40L, which is fused to a sortaseor the active fragment thereof. The first cell may be a B cell, and thesecond cell may be a T cell.

The intercellular labeling method described herein may be performed invitro. Alternatively, it may be performed in vivo. In the latter case,the first cell, the second cell, or both may be endogenous cells of anon-human transgenic animal (i.e., cells produced in the transgenicanimal). Exemplary non-human transgenic animals include, but are notlimited to, transgenic mouse, transgenic rat, or transgenic rabbit. Insome embodiments, the first cell is an endogenous cell of a transgenicanimal, and the second cell is constructed in vitro and transferred intothe same transgenic animal. In other embodiments, the second cell is anendogenous cell of a transgenic animal and the first cell is constructedin vitro and transferred into the same transgenic animal. Alternatively,the in vivo labeling can be performed by constructing the first cell,the second cell, or both in vitro and transferring the cell(s) into asubject, which can be a mouse, a rabbit, a rat, or a monkey.

In any of the intercellular labeling methods described herein, thepeptide comprising the sortase recognition sequence is administered tothe transgenic animal or subject, when the method is performed in vivo.In some embodiments, the contacting step between the first cell and thesecond cell is carried out in a germinal center.

In any of the intercellular labeling methods described herein, thecontacting step can be performed in the presence of a candidatecompound. Such a method may further comprise assessing whether thecandidate compound modulates the interaction between the two members ofthe receptor-ligand pair, wherein a change of the labeling of the firstcell in the presence of the candidate compound indicates that thecompound is a modulator of the receptor-ligand pair. Such a method maybe useful in drug discovery or design.

In another aspect, the present disclosure provides a nucleic acidcomprising a nucleotide sequence that encodes a polypeptide comprising asortase and a member of a ligand-receptor pair, the encoded polypeptide(which may further comprise a protein tag), a vector (e.g., anexpression vector) comprising the nucleic acid, and a host cellcomprising any of the vectors described herein. The sortase, sortaserecognition sequence, and sortase acceptor peptide can be any of thosedescribed herein or know in the art.

In some embodiments, the intercellular labeling method described hereinis applied to determine the antigen specificity of a T cell receptor. Insuch a method, the first cell is an antigen-presenting cell (APC) thatexpresses a MHC class I molecule, a MHC class II molecule, or both; andthe second cell is a T cell that expresses a T cell receptor (TCR).Examples of APCs include, but are not limited to, B cells, dendriticcells, macrophages, or a combination thereof.

In some examples, the APC is engineered to further express a polypeptideencoded by a member of a cDNA library. For example, step (i) may beperformed by providing a plurality of APCs which collectively expresspolypeptides encoded by the cDNA library; and step (iii) may beperformed by contacting the plurality of the APCs with the T cell in thepresence of the sortase substrate. The method may further compriseisolating the labeled APC produced in step (iii). The member of the cDNAlibrary carried by the labeled APC may be identified for determiningantigen specificity of the TCR expressed on the T cell.

In yet another aspect, the present disclosure provides kits forintercellular labeling, comprising: (i) a first cell expressing a firstpolypeptide, which comprises a sortase acceptor peptide (e.g., anoligoglycine as described herein such as G₅), and optionally one memberof a receptor-ligand pair (e.g., those described herein), wherein thesortase acceptor peptide is located at the N-terminus of the firstpolypeptide; and (ii) a second cell expressing a second polypeptide,which comprises a sortase (e.g., any of the sortases disclosed herein,such as a sortase A or a mutant sortase A) or an active fragmentthereof, and optionally the other member of the receptor-ligand pair. Insome instances, the first polypeptide is a fusion polypeptide comprisingboth the sortase acceptor peptide and one member of the receptor-ligandpair. Alternatively or in addition, the second polypeptide is a fusionpolypeptide comprising both the sortase or an active fragment thereofand the other member of the receptor-ligand pair. Optionally, the kitmay further comprise a labeled sortase substrate as described herein.The first polypeptide and/or the second polypeptide may further comprisea protein tag. The first cell, the second cell, or both can be immunecells, such as T cells, B cells, dendritic cells, macrophages, ornatural killer cells. In one example, the first cell is an antigenpresenting cell (e.g., a B cell, a DC, or a macrophage) and the secondcell is a T cell. In another example, the first cell is a T cell and thesecond cell is a B cell.

In one example, the first polypeptide comprises CD40 and oligoglycineGGGGG (SEQ ID NO: 3), which is located at the N-terminus of the firstpolypeptide, and the second polypeptide comprises CD40L, which is fusedto the sortase at the C-terminus. The first cell may be a B cell, andthe second cell may be a T cell.

In another example, the kit is useful in determining TCR specificity,which may comprise (i) a plurality of antigen-presenting cells (APCs)such as B cells, DCs, or macrophages, each expressing one or both of MHCclass I and MHC class II molecules and a polypeptide comprising asortase acceptor peptide, and collectively polypeptides encoded by acDNA library; (ii) a T cell expressing a polypeptide comprising asortase or an active fragment thereof; and optionally (iii) a labeledsortase substrate as described herein.

Also disclosed herein is a non-human animal comprising: (i) a first cellexpressing a first polypeptide, which comprises a sortase acceptorpeptide and optionally one member of a receptor-ligand pair, wherein thesortase acceptor peptide is located at the N-terminus of the firstpolypeptide; (ii) a second cell expressing a second polypeptide, whichcomprises a sortase and optionally the other member of thereceptor-ligand pair, or (i) and (ii). In some examples, the first cell,the second cell, or both are immune cells, including, but not limitedto, T cells, B cells, dendritic cells, macrophages, or natural killercells. In one example, the first cell is a T cell, and the second cellis a B cell, or vice versa.

In some embodiments, the non-human animal is a transgenic animal, inwhich a gene encoding the first polypeptide, a gene encoding the secondpolypeptide, or both are inserted into the genome of the animal.Alternatively, the animal is (a) a transgenic animal, in which a nucleicacid sequence encoding the sortase acceptor peptide is inserted into theendogenous locus encoding the one member of the ligand-receptor pair,leading to the expression of the first polypeptide; or (b) the animal isa transgenic animal, in which a nucleic acid sequence encoding thesortase is inserted into the endogenous locus encoding the other memberof the ligand-receptor pair, leading to the expression of the secondpolypeptide. Examples of transgenic animals include, but are not limitedto, transgenic mouse, rat, or rabbit.

In some examples, the animal is a transgenic animal, in which the geneencoding the first polypeptide is inserted into the genome of theanimal, and the second cell that expresses the second polypeptide can betransferred into the animal. The second cell may be constructed invitro. Alternatively, the animal is a transgenic animal, in which thegene encoding the second polypeptide is inserted into the genome of theanimal, and the first cell that expresses the first polypeptide isconstructed in vitro and transferred into the animal.

In one exemplary non-human animal as described herein, the firstpolypeptide comprises CD40, which is fused to acceptor peptide GGGGG(SEQ ID NO: 3), and the second polypeptide comprises CD40L, which isfused to the sortase at the C-terminus. The first cell may be a B celland the second cell may be a T cell.

In some embodiments, the non-human animal can be a transgenic non-humanmammal (e.g., a transgenic mouse or transgenic rat) that comprises oneor more human immunoglobulin genes or a portion thereof. In otherembodiments, the non-human mammal comprises a humanized immune system.

Further, the present disclosure provides methods for identifying a Bcell expressing a high affinity B cell receptor (BCR) to an antigen, themethod comprising: (i) providing a mammal that comprises (a) a pluralityof B cells expressing a first polypeptide, which comprises a sortaseacceptor peptide, wherein the sortase acceptor peptide is located at theN-terminus of the first polypeptide, and (b) a plurality of T cellsexpressing a second polypeptide, which comprises a sortase or an activefragment thereof; (ii) administering to the animal an effective amountof a peptide comprising a sortase recognition sequence, wherein thepeptide is associated with a detectable label; (iii) isolatinglymphocytes from a germinal center of the animal; and (iv) identifying aB cell that is conjugated to the detectable label, wherein the B cellthus identified expresses a high affinity BCR to an antigen. The animalmay be immunized with an antigen of interest. In some examples, thefirst polypeptide comprises the sortase acceptor peptide fused to amember of a receptor-ligand pair, the member being a B cell protein(i.e., a protein expressed in naturally-occurring B cells).Alternatively or in addition, the second polypeptide comprises thesortase or the active fragment thereof fused to the other member of thereceptor-ligand pair, the other member being a T cell protein (i.e., aprotein expressed in naturally-occurring T cells).

In some examples, the mammal is a transgenic mammal, in which a geneencoding the first polypeptide, a gene encoding the second polypeptide,or both are inserted into the genome of the mammal. In other examples,the mammal is a transgenic animal, in which a nucleic acid sequenceencoding the sortase acceptor peptide is inserted into the endogenouslocus encoding the one member of the ligand-receptor pair, leading tothe expression of the first polypeptide. Alternatively, the mammal is atransgenic animal, in which a nucleic acid sequence encoding the sortaseis inserted into the endogenous locus encoding the other member of theligand-receptor pair, leading to the expression of the secondpolypeptide.

In some examples, the mammal is a transgenic mammal, in which the geneencoding the first polypeptide is inserted into the genome of the mammaland expressed on naïve B cells and the plurality of T cells thatexpresses the second polypeptide, which may be constructed in vitro, aretransferred into the mammal.

Examples of transgenic animals include transgenic mouse, transgenic rat,or transgenic rabbit. Exemplary receptor-ligand pairs include CD40 andCD40L, CD80 and CD28, CD80 and CTLA4, CD86 and CD28, CD86 and CTLA4,PD-1 and PD-L1, PD-1 and PD-L2, and ICOS and ICOSL. In one example, thetransgenic animal is a transgenic mouse expressing a fusion proteincomprising CD40L and SrtA. The nucleotide sequence encoding the SrtA maybe inserted downstream of the last coding exon of the endogenous CD40Lgene. In another example, the transgenic animal is a transgenic mouseexpressing a fusion protein comprising a G5 fragment and CD40. Thenucleotide sequence encoding the five glycine fragment may be insertedin the second exon of the endogenous CD40 gene.

The methods described herein may further comprise isolating one or morenucleic acid encoding at least a portion of a heavy chain variableregion, at least a portion of a light chain variable region, or both ofthe B cell receptor (BCR) from the B cell that is conjugated to thedetectable label. The at least a portion of the heavy chain variableregion, the at least a portion of the light chain variable region, orboth encode at least one complementarity determining region of the BCR.Further, the method may also comprise sequencing the at least a portionof the heavy chain variable region, the at least a portion of the lightchain variable region, or both.

Any of the methods described herein may further comprise producing ahybridoma cell derived from the B cell that is conjugated to thedetectable label, wherein the hybridoma cell produces high affinityantibodies to the antigen. The hybridoma cell thus produced can becultured in vitro for producing the high affinity antibodies.

In some embodiments, the non-human mammal is a transgenic non-humanmammal that comprise human immunoglobulin genes. Examples of suchnon-human mammals include transgenic mice or transgenic rats.Alternatively, the non-human mammal may comprise a humanized immunesystem.

Moreover, the present disclosure provides a method for identifying abinding partner of a protein of interest, which may be performed invitro. Such a method comprising: (i) providing a first population ofcells expressing a plurality of polypeptides, each of the cellsexpressing on its surface a sortase acceptor peptide and a candidateprotein, (ii) providing a second population of cells expressing apolypeptide, the cells expressing on the cell surface a sortase, or anactive fragment thereof, and the protein of interest; (iii) contactingthe first population of cells with the second population of cells in thepresence of a peptide comprising a sortase recognition sequence, whereinthe peptide is associated with a detectable label; (iv) detectinglabeling of cells in the first population of cells; and (v) identifyinga binding partner of the protein of interest, wherein a candidateprotein is a binding partner of the protein of interest, if the cellthat expresses a polypeptide comprising the candidate protein is labeledin step (iii). The polypeptide comprising the protein of interest, thepolypeptides comprising the candidate proteins, or both may furthercomprise a protein tag. In some examples, the sortase acceptor peptideand the protein candidate may be covalently linked to form a fusionpolypeptide. Alternatively or in addition, the sortase or the activefragment thereof and the protein of interest are covalently linked toform a fusion polypeptide.

In some examples, the protein of interest is a receptor of an immunecell, e.g., a T cell, a B cell, dendritic cells, macrophages, or naturalkiller cells. In some examples, the detectable label is biotin or afluorescent dye.

In any of the methods described herein, the sortase can be a sortase A,a sortase B, a sortase C, or a sortase D. In some examples, the sortaseis a mutant SrtA that exhibits improved catalytic activity as comparedto the wild-type counterpart, e.g., those described herein.Alternatively or in addition, the sortase recognition sequence is LPXTG(SEQ ID NO: 1), in which X is any amino acid residue. In one example,the sortase recognition sequence is LPETG (SEQ ID NO: 2), which may beco-used with a mutant SrtA as described herein. Alternatively or inaddition, the sortase acceptor peptide is an oligoglycine, which mayconsist of 1-5 glycine residues.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of intercellular labeling strategy. (A)a schematic illustration showing an exemplary model of SrtA mediatedlabeling strategy applied to CD40/CD40L ligand/receptor pair. (i) CD40Lhas been fused to SrtA while its partner, CD40, has been functionalizedwith five glycine residues. Biotinylated or fluorescently labeled SrtAsubstrate will bind to the enzyme and form a covalent intermediate. (ii)Upon CD40-CD40L interaction, spatial proximity allows the labeledsubstrate to be transferred to the N-terminus glycine of CD40. (iii) Acell carrying G5-CD40 maintains the label after CD40L interaction. (B)Kinetic parameters of CD40-CD40L interaction (Kd) and SrtA mediatedtranspeptidation (KmLPETG (SEQ ID NO: 2); KmGGG). Sequences, from leftto right and top to bottom, correspond to SEQ ID NOs.: 2, 54, and 55.

FIG. 2. Intercellular labeling mediated by CD40/CD40L interaction. (A) Aschematic representation of the constructs employed in this experiment.(B) Graphs showing expression of SrtA-fusion constructs in HEK293T.HEK293T cells were transfected with the constructs listed in (A). Cellscarrying G5-CD40 construct (GFP reporter vector) were incubated withcells transfected with different SrtA-fusion constructs (tdTomatoreporter vector), treated with biotinylated SrtA substrate, stained withstreptavidin, and analyzed by flow cytometry. Dot plot of arepresentative sample shows tdTomato⁺ gating. Histogram plot showsbinding of SrtA substrate to SrtA-fusion construct transfected cells(constructs are color-coded as in panel A; grey peak corresponds tountransfected cells). (C-D) Intercellular labeling mediated byCD40/CD40L interaction in HEK293T cells. Cells were transfected andtreated as in (B). Dot plot of a representative sample shows GFP⁺ (C) orGFP^(int) (D) gating. Histogram plots shows labeling of gated G5-CD40⁺cells by the different SrtA-fusion construct transfected cells. (E)G₅-CD40 specificity of intercellular labeling mediated by CD40/CD40Linteraction. HEK293T cells were transfected with the constructs listedin (A). Cells carrying G₅-CD40 construct were incubated with cellstransfected with different SrtA-fusion constructs, treated withbiotinylated SrtA substrate, and analyzed by western blot withstreptavidin or antibodies specific for Myc tag (G₅-CD40 construct),FLAG tag (SrtA-fusion constructs) and α-tubulin.

FIG. 3. Intercellular labeling in primary murine lymphocytes. (A) Aschematic representation of the cells and constructs employed in thisexperiment. Primary murine B and CD4⁺ T lymphocytes were purified fromC57BL/6 CD40L^(−/−) mice and CD40L^(−/Y) TCR-transgenic OT-II mice,respectively. CD4⁺ T cells were transduced with retroviral vectorscarrying CD40L-SrtA, CD40L^(K142E-R202E)-SrtA (both tdTomato reportervector) or left untransduced. B cells were transduced with G5-CD40 (GFPreporter vector). (B) Expression of SrtA constructs in CD0⁺ T cells. Band CD4⁺ T lymphocytes were co-cultured in the presence or absence ofOVA³²³⁻³³⁹ peptide, treated with biotin-LPETGG (SEQ ID NO: 5) andstained with streptavidin before flow cytometry analysis. Dot plot showsgating of tdTomato⁺ cells while histogram plot show binding ofbiotinylated substrate on gated SrtA expressing cells. (C) Intercellularlabeling of G₅-CD40 B cells. Cells co-cultured and treated as in (B)were gated based on GFP expression to identify B cells carrying G5-CD40construct (left dot-plot). Intensity of labeling in GFP⁺ gated cellsco-cultured with or without OVA³²³⁻³³⁹ peptide is displayed on histogramplot.

FIG. 4. Intercellular labeling mediated by various ligand-receptor pairinteraction. (A) A schematic representation of the constructs employedin this experiment. (B) Cells carrying G₅-fusion constructs (GFPreporter vector) were incubated with cells transfected with differentSrtA-fusion constructs (tdTomato reporter vector), treated withbiotinylated SrtA substrate, stained with streptavidin and analyzed byflow cytometry. Each G₅-fusion construct (G₅-CD80, G₅-CD86, G₅-PD-L1,G₅-PD-L2, G₅-ICOSL) has been matched with the interacting partner(s),SrtA-PDGFR or untransfected cells. Dot plot of a representative sampleshows GFP⁺ gating. Histogram plots shows labeling of gated G-fusionconstruct cells by different SrtA-fusion construct transfected cells.(C) Expression of SrtA-fusion constructs in HEK293T. Cells as in (B)were gated for tdTomato expression as in the dot plot to identifySrtA-expressing cells. Histogram plot shows binding of SrtA substrate toSrtA-fusion constructs.

FIG. 5. Delivery of SrtA substrate and T cell labeling in vivo. (A) Aschematic representation of the experimental setup. CD4⁺ T cells werepurified from C57BL/6 GFP⁺ mice and transduced with either SrtA-PDGFR(tdTomato reporter vector) or tdTomato only as a control. 1.5×10⁶transduced CD4⁺ T cells were transferred intravenously in recipientC57BL/6 and 250 nmol of biotin-LPETGG (SEQ ID NO: 5) injectedsubcutaneously at the base of the tail 24 hours after T cell transfer.Inguinal lymph nodes were harvested 1 hour after biotin-LPETGG (SEQ IDNO: 5) injection and analyzed by flow cytometry. (B) A schematicrepresentation of the construct used in this experiment. (C) In vivolabeling of SrtA-expressing T cells. Dot plots show subsequent CD4⁺ andGFP⁺ gating to identify transferred CD4⁺ T cells. Labeling intensityversus tdTomato expression is shown in dot plots on the right.

FIG. 6. G₅-CD40 or G₅-CD86 knock-in mice and labeling of cellsexpressing G₅-CD40 in the knock-in mice treated with a biotinylated SrtAsubstrate and SrtA. Left panel: a schematic illustration of the knock-inmice. Middle panel: a diagram showing sequencing results that confirminsertion of transgenes. Right panel: a diagram showing the labeling oflymphocytes isolated from G₅-CD40 knock-in mice treated withbiotinylated SrtA substrate and 3 μM SrtA. Sequences, from top tobottom, correspond to SEQ ID NOs.: 56 and 57.

FIG. 7. Intercellular labeling in primary murine lymphocytes. Primarymurine B and CD4+T lymphocytes were purified from C57BL/6 mice andTCR-transgenic OT-II mice, respectively. CD4+ T cells were transducedwith retroviral vectors carrying CD40L-SrtA or SrtA-PDGFR or leftuntransduced. B cells were transduced with G5-CD40. B and CD4+Tlymphocytes were co-cultured in presence or absence of OVA323-339peptide, treated with biotin-LPETGG (SEQ ID NO: 5) and stained withstreptavidin before flow cytometry analysis. Histogram plots showlabeling of G5-CD40 B cells.

FIG. 8. Generation of CD40L-SrtA gene-targeted mouse. (A) Targetingstrategy. (B) Southern blot analysis of CD40L-SrtA targeted mouse.

FIG. 9. Generation of G5-CD40 gene-targeted mouse. (A) Targetingstrategy. Sequences from top to bottom correspond to SEQ ID NOs.: 58 and59. (B) Restriction fragment length polymorphism analysis of targetedanimal. (C) Sequencing results of the insertion region in the targetedanimal. Sequence corresponds to SEQ ID NO: 60.

FIG. 10. Intercellular labeling upon B cell: CD4+ T cell interaction exvivo. (A) Schematic representation of the experimental set-up. (B)Formation of Biotin-LPET:SrtA (SEQ ID NO: 54) covalent intermediate inCD40L-SrtA+/Y OT-II CD4+ T cells upon interaction withantigen-presenting B cells and dot-plot and histogram plot ofG5-CD40+/+B cells.

FIG. 11. Intercellular labeling upon DC:CD4+ T cell interaction in vivo.(A) Schematic representation of the experimental set-up. C57BL/6 micewere injected subcutaneously (s.c.) in the footpad with 1-2×106OVA323-339 pulsed DCs/footpad. 24 hours later, mice were injectedintravenously (i.v.) with 5-10×106 CD40L-SrtA+/Y or −/Y OT-II CD4+ Tcells. 15 hours after T cell transfer, mice were injected s.c. with 1umol of Biotin-LPETGG (SEQ ID NO: 5) every 30 min for a total of 4hours. Popliteal LNs were then harvested and analyzed by flow cytometry.(B) Formation of Biotin-LPET:SrtA (SEQ ID NO: 54) covalent intermediatein CD40L-SrtA+/Y OT-II CD4+ T cells. (C) Labeling in the DCs population.Labeling is specifically detectable in antigen-pulsed DCs.

DETAILED DESCRIPTION OF THE INVENTION

Effective humoral immune response requires the production of antibodiesthat bind with high affinity and specificity to the antigen. Thegeneration of high-affinity antibodies takes place in germinal centers(GCs), specialized anatomic structures within lymph nodes. There, Bcells exhibiting randomly mutated immunoglobulin molecules (BCRs) at theplasma membrane are selected based on their affinity for the antigen.Recent studies on the cellular mechanism mediating affinity-basedselection of B cells revealed a key role of the interaction between Bcells and follicular T helper cells (Tfh). Despite the emerging role ofthis interaction in the generation of high-affinity antibody, themolecular events beyond this process are poorly characterized.

The present disclosure is based, at least in part, on the development ofa novel approach to track interactions between cells, such as immunecells, in vitro (e.g., using cell lines), ex vivo (e.g., using primarymurine lymphocytes), and/or in vivo (e.g., in mice), based on theenzymatic labeling of receptor and ligand molecules oriented across theimmunological synapse. The present study has achieved intercellularlabeling between cultured T cells and B cells in vitro, primary murine Band T cells ex vitro, and B cells and T cells in vivo. It was alsodemonstrated that the labeling occurs upon interaction between severalligand-receptor pairs, and that labeling intensity depends onligand-receptor affinity; labeling between non interacting moleculesoccurs at low levels, in the context of cognate interaction. By celltransfer, it has been demonstrated that enzymatic activity required forintercellular labeling is maintained in vivo, and that enzyme substratecan easily delivered to secondary lymphoid organs. This system wouldallow for determining the molecular signature triggered in B cells bythe interaction with Tfh cells in vivo and to identify the key factorsand pathways that control affinity-based selection of B cells in the GC.Moreover, this system has the potential as a broadly useful tool fortracking cell-cell interactions in vivo that can be applied to mostbiological areas. Further, the intercellular labeling system describedherein can be applied to identify particular antigens to which a T cellreceptor of unknown specificity responds. This would be useful toidentify the particular antigens that stimulate T cell responses invarious diseases, such as cancer, infection, and autoimmune diseases.One could, for example, isolate T cells from a disease site, e.g., acancer site, a site of infection, or an affected site of an autoimmunedisease, and identify the antigens to which the involved T cellsrespond. The antigens thus identified might be useful as, e.g., vaccineantigens.

Accordingly, described herein are intercellular labeling methods anduses of such labeling methods to track ligand-receptor interactions bothin vitro and in vivo, to identify compounds capable of modulatingligand-receptor interaction, to identify potential binding partners ofproteins of interest, or to identify cognate antigens for TCRs withunknown specificities.

I. Intercellular Labeling Mediated by Ligand-Receptor Interaction

The intercellular labeling method described herein involves two types ofengineered cells. The first type of cell is engineered to express on thecell surface a polypeptide comprising a sortase or an active fragmentthereof. The second type of cell is engineered to express on the cellsurface a polypeptide comprising a sortase acceptor peptide (e.g.,oligoglycine), which is located at the N-terminus of the polypeptide.These two cells can interact with each other via, e.g., areceptor-ligand pair expressed on the surface of the cells, to bring thetwo cells together. The sortase acceptor peptide and/or thesortase/active fragment thereof may or may not be fused to the receptorand ligand, respectively. Upon interaction, the spatial proximity of thetwo cells would allow for the sortase or the active fragment thereofexpressed on the second type of cell to transfer a sortase substrateonto the sortase acceptor expressed on the first type of cell.Accordingly, the two cells can be incubated in the presence of a labeledpeptide comprising a sortase recognition sequence, which binds to thepolypeptide comprising a sortase expressed on the surface of one cell,to label the cell expressing the sortase acceptor peptide. A schematicillustration of this intercellular labeling method is provided in FIG.1.

(i) Sortase-Mediated Cell Surface Labeling

Described herein are sortase-mediated cell surface labeling methods,which allow for tracking molecule interactions both in vitro and invivo.

(a) Sortase

Sortases are a family of enzymes capable of carrying out atranspeptidation reaction conjugating the C-terminus of a protein to theN-terminus of another protein via transamidation. Sortases are alsoreferred to as transamidases, and typically exhibit both a protease anda transpeptidation activity. Various sortases from prokaryotic organismshave been identified. For example, some sortases from Gram-positivebacteria cleave and translocate proteins to proteoglycan moieties inintact cell walls. Among the sortases that have been isolated fromStaphylococcus aureus, are sortase A (Srt A) and sortase B (Srt B).Thus, in certain embodiments, a transamidase used in accordance with theintercellular labeling methods described herein is a sortase A, e.g.,from S. aureus, also referred to herein as SrtAaureus. In otherembodiments, a transamidase is a sortase B, e.g., from S. aureus, alsoreferred to herein as SrtBaureus.

Sortases have been classified into four classes, designated A, B, C, andD (i.e., sortase A, sortase B, sortase C, and sortase D, respectively)based on sequence alignment and phylogenetic analysis of 61 sortasesfrom Gram-positive bacterial genomes (Dramsi et al., Res Microbiol.156(3):289-97, 2005; the entire contents of which are incorporatedherein by reference). These classes correspond to the followingsubfamilies, into which sortases have also been classified by Comfortand Clubb (Comfort et al., Infect Immun., 72(5):2710-22, 2004; theentire contents of which are incorporated herein by reference): Class A(Subfamily 1), Class B (Subfamily 2), Class C (Subfamily 3), and Class D(Subfamilies 4 and 5). The aforementioned references disclose numeroussortases and their recognition motifs. See also Pallen et al., TRENDS inMicrobiology, 2001, 9(3), 97-101; the entire contents of which areincorporated herein by reference). Those skilled in the art will readilybe able to assign a sortase to the correct class based on its sequenceand/or other characteristics such as those described in Drami, et al.,supra.

The term “sortase A” is used herein to refer to a class A sortase,usually named SrtA in any particular bacterial species, e.g., SrtA fromS. aureus. Likewise “sortase B” is used herein to refer to a class Bsortase, usually named SrtB in any particular bacterial species, e.g.,SrtB from S. aureus. The present disclosure encompasses embodimentsrelating to any of the sortase classes known in the art (e.g., a sortaseA from any bacterial species or strain, a sortase B from any bacterialspecies or strain, a class C sortase from any bacterial species orstrain, and a class D sortase from any bacterial species or strain).

In some embodiments, the sortase used in the intercellular labelingmethods described herein is a wild-type enzyme. In other embodiments,the sortase is a modified version which may possess a superior featureas compared to the wild-type counterpart (e.g., higher catalyticactivity). In some examples, the sortase can be a mutant of SrtA, whichmay comprise one or more of the following positions: P94, S102, A104,E105, K138, K152, D160, K162, T164, D165, K173, 1182, K190, and K196.For example, a SrtA mutant may comprise one or more of the followingmutations: P94R or P94S, S102C, A104H, E105D, K138P, K152I, D160K orD160N, K162H, T164N, D165A, K173E, I182V, K190E, and K196S or K196T. Inone example, the sortase is a triple mutant P94S/D160N/K196T of SrtAfrom S. aureus.

In other embodiments, modified sortase having altered substratespecificity can be used in the intercellular labeling methods describedherein. For example, sortase A mutants having one or more mutations atpositions S102 (e.g., S102C), A104 (e.g., A104H or A104V), E105 (e.g.,E105D), K138 (e.g., K138P), K152 (e.g., K152I), N162 (e.g., N162N), T164(e.g., T164N), K173 (e.g., K173E), 1182 (e.g., I182V), T196 (e.g.,T196S), N98 (e.g., N98D), A118 (e.g., A118T), F122 (e.g., F122A), K134(e.g., K134R), F144 (e.g., F144L), and E189 (e.g., E189F). Such amodified sortase may recognize sequences such as LAXTG (SEQ ID NO: 6)and/or LPXSG (SEQ ID NO: 7), in which X can be any amino acid residue.Examples include mutantS102C/A104H/E105D/K138P/K152I/N162N/T164N/K173E/I182V/T196S, and mutantN98D/A104V/A118T/F122A/K134R/F144L/E189F. Additional sortase mutantshaving altered substrate specificity are disclosed in US20140057317 andDorr et al., PNAS 111 (37): 13343-13348 (2014), the relevant disclosurestherein are incorporated by reference herein.

A modified version of a wild-type sortase may share at least 85% (e.g.,90%, 95%, 98%, or above) sequence identity to the wild-type counterpart.It may contain mutations at one or more positions corresponding to thosedescribed above, which can be identified by analyzing the amino acidsequence of a wild-type sortase with the amino acid sequence of a SrtA.The “percent identity” of two amino acid sequences can be determinedusing the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of the invention. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

In some embodiments, the intercellular labeling methods can use anactive fragment of a sortase. Such a fragment of a specific sortase canbe identified based on knowledge in the art or by comparing the aminoacid sequence of that sortase with a sortase having knownstructure/function correlation (e.g., active domain being identified).In some examples, the sortase used herein can be an active fragment of asortase A such as SrtA from S. aureus, e.g., a sortase A fragmentlacking the N-terminal 59 or 60 amino acid residues, or a functionalvariants thereof, which may contain one or more of the mutationsdescribed herein.

Amino acid sequences of Srt A and Srt B and the nucleotide sequencesthat encode them are known to those of skill in the art and aredisclosed in a number of references cited herein, the entire contents ofall of which are incorporated herein by reference. See, e.g., GenBankaccession numbers NP_375640 and YP_043193. The amino acid sequences ofS. aureus SrtA and SrtB are homologous, sharing, for example, 22%sequence identity and 37% sequence similarity. The amino acid sequenceof a sortase-transamidase from Staphylococcus aureus also hassubstantial homology with sequences of enzymes from other Gram-positivebacteria, and such transamidases can be utilized in the ligationprocesses described herein. For example, for SrtA there is about a 31%sequence identity (and about 44% sequence similarity) with bestalignment over the entire sequenced region of the S. pyogenes openreading frame. There is about a 28% sequence identity with bestalignment over the entire sequenced region of the A. naeslundii openreading frame. It will be appreciated that different bacterial strainsmay exhibit differences in sequence of a particular polypeptide, and thesequences herein are exemplary.

In certain embodiments a transamidase bearing 18% or more sequenceidentity, 20% or more sequence identity, or 30% or more sequenceidentity with an S. pyogenes, A. naeslundii, S. mutans, E. faecalis orB. subtilis open reading frame encoding a sortase can be screened, andenzymes having transamidase activity comparable to Srt A or Srt B fromS. aureus can be utilized (e.g., comparable activity sometimes is 10% ofSrt A or Srt B activity or more).

In some embodiments, the intercellular labeling methods described hereinuse a sortase A (SrtA) or an active fragment thereof. SrtA recognizesthe motif LPXTX; wherein each occurrence of X represents independentlyany amino acid residue), with common recognition motifs being, e.g.,LPKTG (SEQ ID NO: 8), LPATG (SEQ ID NO: 9), or LPNTG (SEQ ID NO: 10). Insome embodiments LPETG (SEQ ID NO: 2) is used as the sortase recognitionmotif. However, motifs falling outside this consensus may also berecognized. For example, in some embodiments the motif comprises an ‘A’rather than a ‘T’ at position 4, e.g., LPXAG (SEQ ID NO: 11), or LPNAG(SEQ ID NO: 12). In some embodiments the motif comprises an ‘A’ ratherthan a ‘G’ at position 5, e.g., LPXTA (SEQ ID NO: 13), or LPNTA (SEQ IDNO: 14). In some embodiments the motif comprises a ‘G’ rather than ‘P’at position 2, e.g., LGXTG (SEQ ID NO: 15) or LGATG (SEQ ID NO: 16). Insome embodiments the motif comprises an ‘I’ rather than ‘L’ at position1, e.g., IPXTG (SEQ ID NO: 17), IPNTG (SEQ ID NO: 18) or IPETG (SEQ IDNO: 19). Additional suitable sortase recognition motifs will be apparentto those of skill in the art, and the invention is not limited in thisrespect. It will be appreciated that the terms “recognition motif” and“recognition sequence”, with respect to sequences recognized by atransamidase or sortase, are used interchangeably. In some embodiments,the SrtA is a mutant as described herein, which may possess improvedenzymatic activity relative to the wild-type counterpart. Such a mutantmay recognize LAETG (SEQ ID NO: 20) and use a peptide comprising therecognition sequence as a substrate. Such sortase recognition motifs canbe used in any of the methods described herein.

In some embodiments of the invention the sortase is a sortase B (SrtB)or an active fragment thereof, e.g., a sortase B of S. aureus, B.anthracis, or L. monocytogenes. Motifs recognized by sortases of the Bclass (SrtB) often fall within the consensus sequences NPXTX, e.g.,NP[Q/K]-[T/s]-[N/G/s] (SEQ ID NO: 21), such as NPQTN (SEQ ID NO: 22) orNPKTG (SEQ ID NO: 23). For example, sortase B of S. aureus or B.anthracis cleaves the NPQTN (SEQ ID NO: 22) or NPKTG (SEQ ID NO: 23)motif of IsdC in the respective bacteria (see, e.g., Marraffini et al.,Journal of Bacteriology, 189(17): 6425-6436, 2007). Other recognitionmotifs found in putative substrates of class B sortases are NSKTA (SEQID NO: 24), NPQTG (SEQ ID NO: 25), NAKTN (SEQ ID NO: 26), and NPQSS (SEQID NO: 27). For example, SrtB from L. monocytogenes recognizes certainmotifs lacking P at position 2 and/or lacking Q or K at position 3, suchas NAKTN (SEQ ID NO: 26) and NPQSS (SEQ ID NO: 27) (Mariscotti et al., JBiol Chem. 2009 Jan. 7). Such sortase recognition motifs can also beused in any of the methods described herein.

Using sortases with distinct substrate specificity, it is possible tocombine N-terminal and C-terminal labeling strategies (Antos et al.,2009, J. Am. Chem. Soc., 131(31):10800-10801) to generate multi-labeledcells. For example, unlike Sortase A from Staphylococcus aureus, SortaseA derived from Streptococcus pyogenes recognizes LPXTA (SEQ ID NO: 13)motifs and accepts oligo-alanine probes as nucleophiles. Therefore, thesortase reactions of both enzymes can be performed as orthogonalreactions. Utilization of such sortase reactions with suitablesortase(s) is also within the scope of the present disclosure.

In some embodiments, the sortase is a sortase C (Srt C) or an activefragment thereof. Sortase C may utilize LPXTX as a recognition motif,with each occurrence of X independently representing any amino acidresidue. This recognition motif can be used for constructing thesortaggable surface proteins described herein.

In yet other embodiments, the sortase is a sortase D (Srt D) or anactive fragment thereof. Sortases in this class are predicted torecognize motifs with a consensus sequence NA-[E/A/S/H]-TG (SEQ ID NO:28; Comfort D, supra). Sortase D has been found, e.g., in Streptomycesspp., Corynebacterium spp., Tropheryma whipplei, Thermobifida fusca, andBifidobacterium longhum. LPXTA (SEQ ID NO: 13) or LAXTG (SEQ ID NO: 6)may serve as a recognition sequence for sortase D, e.g., of subfamilies4 and 5, respectively subfamily-4 and subfamily-5 enzymes process themotifs LPXTA (SEQ ID NO: 13) and LAXTG (SEQ ID NO: 6), respectively. Forexample, B. anthracis Sortase C has been shown to specifically cleavethe LPNTA (SEQ ID NO: 14) motif in B. anthracis BasI and BasH (seeMarrafini, supra).

Additional sortases and their active fragments, including, but notlimited to, sortases recognizing additional sortase recognition motifsare also suitable for use in some embodiments of this invention. Forexample, sortases described in Chen et al., Proc Natl Acad Sci USA. 2011Jul. 12; 108(28):11399 (the entire contents of which are incorporatedherein); and a sortase that recognizes QVPTGV (SEQ ID NO: 29) motif asdescribed in Barnett et al., Journal of Bacteriology, Vol. 184, No. 8,p. 2181-2191, 2002 (the entire contents of which are incorporated hereinby reference).

The use of sortases found in any gram-positive organism, such as thosementioned herein and/or in the references (including databases) citedherein is contemplated in the context of some embodiments of thisinvention. Also contemplated is the use of sortases found in gramnegative bacteria, including, but not limited to, Colwelliapsychrerythraea, Microbulbifer degradans, Bradyrhizobium japonicum,Shewanella oneidensis, and Shewanella putrefaciens. Such sortasesrecognize sequence motifs outside the LPXTX consensus, for example,LP[Q/K]T[A/S]T (SEQ ID NO: 30). In keeping with the variation toleratedat position 3 in sortases from gram-positive organisms, a sequence motifLPXT[A/S] (SEQ ID NO: 31), e.g., LPXTA (SEQ ID NO: 13) or LPSTS (SEQ IDNO: 32) may be used.

(b) Sortase-Catalyzed Transpeptidation Reaction

Sortase-catalyzed transacylation reactions, and their use intranspeptidation (sometimes also referred to as transacylation) forprotein engineering are well known to those of skill in the art (see,e.g., Ploegh et al., WO/2010/087994, and Ploegh et al., WO/2011/133704,the entire contents of which are incorporated herein by reference). Ingeneral, the transpeptidation reaction catalyzed by sortase results inthe conjugation of a first protein containing a C-terminal sortaserecognition motif, e.g., LPXTX; wherein each occurrence of Xindependently represents any amino acid residue), with a second proteincomprising an N-terminal sortase acceptor peptide, e.g., one or moreN-terminal glycine residues. In some embodiments, the sortaserecognition motif is a sortase recognition motif described herein. Incertain embodiments, the sortase recognition motif is LPXT or LPXTG (SEQID NO: 1).

The sortase transacylation reaction provides means for efficientlylinking an acyl donor with a nucleophilic acyl acceptor. This principleis widely applicable to many acyl donors and a multitude of differentacyl acceptors. Previously, the sortase reaction has been employed forligating proteins and/or peptides to one another, ligating syntheticpeptides to recombinant proteins, linking a reporting molecule to aprotein or peptide, joining a nucleic acid to a protein or peptide,conjugating a protein or peptide to a solid support or polymer, andlinking a protein or peptide to a label. Such products and processessave cost and time associated with ligation product synthesis and areuseful for conveniently linking an acyl donor to an acyl acceptor.

Sortase-mediated transpeptidation reactions (also sometimes referred toas transacylation reactions) are catalyzed by the transamidase activityof sortase, which forms a peptide linkage (an amide linkage), between anacyl donor compound and a nucleophilic acyl acceptor containing anNH₂—CH₂-moiety.

(ii) Engineered Cells Expressing Sortase-Ligand/SortaseAcceptor-Receptor Fusion Polypeptides

The intercellular labeling methods described herein involve at least twocells, one of which is engineered to express on the surface a sortaseacceptor peptide and the other of which is engineered to express asortase or an active fragment thereof on the surface.

To facilitate surface expression, the sortase acceptor peptide and/orthe sortase/active fragment thereof may be fused to a transmembranedomain, and optionally a hinge domain.

Exemplary transmembrane domains include, but are not limited to, atransmembrane of a single-pass membrane protein, e.g., CD8α, CD8β,4-1BB, CD28, CD34, CD4, FcεRIγ, CD16 (e.g., CD16A or CD16B), OX40, CD3ζ,CD3ε, CD3γ, CD3δ, TCRα, CD32 (e.g., CD32A or CD32B), CD64 (e.g., CD64A,CD64B, or CD64C), VEGFR2, FAS, and FGFR2B. Alternatively, thetransmembrane domain may be a non-naturally occurring hydrophobicprotein segment. The hinge domain may be a hinge domain from CD8α orfrom an immunoglobulin molecule. It is expected that the physicalinteraction will be sufficiently stable and specific to allow forspecific labeling of the cells without need for the sortase and sortaseacceptor peptides to be fused to members of a particular interactingreceptor-ligand pair.

The cells for use in the labeling methods described herein can be anytype of cells capable of expressing proteins on cell surfaces. Examplesinclude, but not limited to, bacterial cells, yeast cells, insect cells,plant cells, avian cells (e.g., chicken cells), and mammalian cells(e.g., mouse, rat, rabbit, camelid, non-human primate, human). The atleast two cells involved in the labeling methods can be of the sametype. Alternatively, they can be different types of cells.

In some embodiments, the cells can be immune cells, including, but notlimited to, T cells, B cells, dendritic cells, macrophages, naturalkiller cells, neutrophils, basophils, monocytes, and eosinophils.Examples of T cells include, but are not limited to, CD4⁺ T cells (alsoknown as T helper cells or Th cells, including T_(H)1, T_(H)2, T_(H)3,T_(H)17, Th9, and follicular T helper cells or Tfh cells), CD8⁺ cells(also known as cytotoxic cells or CTLs), memory T cells, and regulatoryT cells (T_(reg) cells). The B cells used in the method described hereincan be a B cell of any development stage (including progenitor B cells,early pro-B cells, late pro-B cells, large pre-B cells, small pre-Bcells, immature B cells, and mature B cells), or any type of B cells(including plasma B cells, memory B cells, B-1 cells, B-2 cells,marginal-zone B cells, follicular B cells, and regulatory B cells). Inone example, one cell can be a T cell and the other cell can be a Bcell, a dendritic cell, or a macrophage. Alternatively, one cell can bea B cell and the other cell can be a T cell, a dendritic cell, or amacrophage. Additional examples include the combination of T cell/Tcell, T cell/B cell, B cell/B cell, T cell/dendritic cell, Tcell/macrophage cell, B cell/dendritic cell, and B cell/macrophage cell.

In some embodiments, one or both cells are a hematological orhematopoietic cell.

In some embodiments, one or both of the cells can be a diseased cell,for example, a cancer cell, including, but not limited to, a breastcancer cell, a lung cancer cell, a liver cancer cell, a kidney cancercell, an oral cancer cell, a skin cancer cell, a cervical cancer cell,an ovarian cancer cell, a pancreatic cancer cell, a brain tumor cell, amelanoma cell, a colon cancer cell, a leukemia cell, or a lymphoma cell.Alternatively, one or both of the cells can be a normal cell of any typeor derived from any tissue, including, but not limited to, breast cell,lung cell, liver cell, kidney cell, skin cell, cervical cell, ovariancell, pancreatic cell, brain cell, or blood cell.

The interaction between the two engineered cells described herein may bemediated by a receptor-ligand pair, each of which is expressed on thesurface of one of the two engineered cells. The receptor, the ligand, orboth may be endogenous to the engineered cells. Alternatively, they canbe exogenous to the cells. In the latter case, expression vectorscarrying genes encoding the receptor or the ligand can be constructedvia conventional technology and introduced into the cells for surfaceexpression. In some embodiments, the receptor, the ligand, or both areexpressed on the surface of the cells at a high level to improve thestability of the cell-cell interaction. In some embodiments, either orboth members of the receptor-ligand pair are ones whose cell surfaceexpression is upregulated upon a cognate interaction (e.g., upon MHC:TCRrecognition where the MHC molecule displays a peptide recognized by theTCR).

In some embodiments, the polypeptide comprising the sortase acceptorpeptide and a member of the receptor-ligand pair can be expressed on thesurface of the first type of engineered cells as separate polypeptides.The polypeptide comprising the sortase or the active fragment thereofand the other member of the receptor-ligand pair can also be express onthe surface of the second type of cells as separate polypeptides.

In other embodiments, the sortase acceptor peptide and the member of thereceptor-ligand pair can be covalently linked to form a single, fusionpolypeptide, which is expressed on the surface of the first type ofcell. The sortase or the active fragment thereof and the other member ofthe receptor-ligand can also be linked covalently to form a fusionpolypeptide, which is expressed on the surface of the second type ofcell.

As used herein, a “ligand-receptor pair” refers to a pair of molecules(e.g., biological molecules) that have a specific affinity for eachother. One member of the receptor-ligand pair may be localized on thesurface of a cell, and preferably on the surface of the plasma membrane,at some point in its existence in vivo. Within a given receptor-ligandpair, either member may be considered to be the ligand or the receptor.Examples of ligand-receptor pairs include, but are not limited to, acell surface receptor and its ligand, (e.g., an oncogene-encodedreceptor and its ligand, a growth factor and its receptor, such as alymphokine and its receptor or an interleukin and its receptor); anenzyme and its substrate; an enzyme and a specific inhibitor or othernon-catalyzable substrate of the enzyme; a hormone and its receptor; afirst subunit of a multimeric protein and a second subunit of themultimeric protein, (for example, two subunits of an immunoglobulinmolecule); a polypeptide portion of a protein and a non-peptide cofactorof the protein; a molecule involved in cellular adhesion, such as acarbohydrate involved in cell adhesion; a cadherin; a cell adhesionmolecule (CAM) (e.g., cell-CAM, neural N-CAM, or muscle N-CAM); alaminin; a fibronectin; or an integrin and the molecule to which itbinds, which may or may not be a cellular adhesion molecule; a firstcomponent of an organelle, the mitotic or meiotic apparatuses, or othersubcellular structure, that displays a specific interaction with asecond component of the same structure or a related structure; a lectinand a carbohydrate; a toxin and its receptor (e.g., diphtheria toxin andits cell surface receptor); a component of a virus and its cell surfacereceptor; or, an IgE molecule and an IgE receptor (e.g., the IgEreceptor found on mast cells, or any other Ig molecule and itsreceptor).

In some embodiments, a ligand-receptor pair used in the intercellularlabeling methods described herein are naturally-occurringligand-receptor pair. Alternatively, one or both of the members of aligand-receptor pair may be a modified version of a naturally-occurringmolecule; the modified version may have improved or decreased bindingactivity to the other member of the pair.

In some examples, the ligand-receptor pair used in the labeling methodsdescribed herein can be a ligand-receptor pair expressed on immunecells. For example, the ligand-receptor pair can be a T cellco-stimulatory molecule and its ligand, or a B cell receptor and itsligand. Examples include, but are not limited to, CD28/CD80, CD28/CD86,CTLA4/CD80, CTLA4/CD86, CD40/CD40L, PD-1/PD-L1, PD-1/PD-L2, ICOS/ICOSL,OX40/OX40L, CD27/CD27L, and 4-1BB/4-1BBL. Preferably, at least one ofthe two members of a ligand-receptor pair has its N-terminus exposed tothe extracellular or luminal space (e.g., a Type I membrane protein).

CD28 is expressed on T cells (on most CD4⁺ T cells and some CD8⁺ Tcells) and CTLA4 (also known as CD152) is usually expressed on activatedT cells. Their ligands, CD80 and CD86 (also known as B7-1 and B7-2) areusually expressed on antigen presenting cells such as dendritic cells, Bcells, and macrophage cells. CD40L (also known as CD154) is expressed onactivated T cells and its binding partner, CD40, is expressed on Bcells, dendritic cells, macrophages, and endothelial cells. ICOS (alsoknown as CD278) is expressed on activated T cells and its ligand, ICOSL(also known as CD275) is expressed on antigen presenting cells such as Bcells, dendritic cells, macrophages, and endothelial cells. Other immunecell ligand-receptor pairs and the type of immune cells on which theyexpress are known in the art. See, e.g., Abbas et al., Cellular andMolecular Immunology, 4^(th) ed., W.B. Saunders Co. (the relevantdisclosures therein are incorporated by reference herein).

Members of the ligand-receptor pair used in the labeling methodsdescribed herein can be full-length proteins as they exist in nature.Alternatively, one or both members of the ligand-receptor pair can be afragment of the naturally-occurring ligand-receptor, which may comprisethe extracellular domain involved in interacting with its bindingpartner.

To perform the intercellular labeling methods described herein, onemember of any of the ligand-receptor pair as described herein, which hasits N-terminus exposed to the extracellular or luminal space, can befused to a sortase acceptor peptide, which is located at the N-terminusof the fusion polypeptide, via methods known in the art, e.g.,recombinant technology. A sortase acceptor peptide can be any peptidethat provides a nucleophilic acyl group for accepting a sortasesubstrate (a peptide comprising a sortase recognition sequence asdescribed herein). Such an acceptor peptide may contain up to about 50amino acids, such as up to 40, 30, 20, 15, 10, or 5 amino acids. In someembodiments, the acceptor peptide is an oligoglycine or oligoalanine,such as a 1-5 glycine fragment or a 1-5 alanine fragment. In someexamples, the oligoglycine consists of 3 or 5 glycine residues. In otherexamples, the oligoalanine consists of 3 or 5 alanine residues.

The other member of the ligand-receptor pair can be fused to a suitablesortase, e.g., a sortase capable of transferring its substrate onto thesortase acceptor peptide fused to the binding partner. For example, aSrtA of S. aureus can be used when the corresponding acceptor peptide isan oligoglycine. Alternatively, a SrtA of S. pyogenes can be used whenthe corresponding acceptor peptide is an oligoalanine. In some examples,the sortase is fused to the N-terminus of the member of theligand-receptor pair. In other examples, the sortase is fused to theC-terminus of the member of the ligand-receptor pair.

In some embodiments, one or both of the fusion polypeptides describedherein may further comprise a protein tag, which may be useful forpurifying, expressing, solubilizing, and/or detecting a polypeptide. Aprotein tag may be relatively small, e.g., ranging from a few aminoacids up to about 100 amino acids long. Alternatively, a protein tag maybe more than 100 amino acids long, e.g., up to about 500 amino acidslong, or more. The use of protein tags in recombinant technology is wellknown in the art. Exemplary protein tags include, but are not limitedto, an HA, TAP, Myc, 6×His, Flag, streptavidin, biotin, or GST tag, toname a few examples. In some embodiments, a protein tag is asolubility-enhancing tag (e.g., a SUMO tag, NUS A tag, SNUT tag, or amonomeric mutant of the Ocr protein of bacteriophage T7). See, e.g.,Esposito D and Chatterjee D K. Curr Opin Biotechnol.; 17(4):353-8(2006). If desired, a protein tag can be cleavable so that it can beremoved, e.g., by a protease. In some embodiments, this is achieved byincluding a protease cleavage site in the tag, e.g., adjacent or linkedto a functional portion of the tag. Exemplary proteases include, e.g.,thrombin, TEV protease, Factor Xa, and PreScission protease. In someembodiments, a “self-cleaving” tag is used. See, e.g., WO/2005/086654.

Nucleic acids encoding the fusion polypeptides described herein, can beinserted into a suitable vector (e.g., a retroviral vector) usingmethods well known in the art. Sambrook et al., Molecular Cloning, ALaboratory Manual, 3^(rd) Ed., Cold Spring Harbor Laboratory Press. Forexample, the gene and vector can be contacted, under suitableconditions, with a restriction enzyme to create complementary ends oneach molecule that can pair with each other and be joined together witha ligase. Alternatively, synthetic nucleic acid linkers can be ligatedto the termini of a gene. These synthetic linkers contain nucleic acidsequences that correspond to a particular restriction site in thevector. Additionally, the vector can contain, for example, some or allof the following: a selectable marker gene, such as the neomycin genefor selection of stable or transient transfectants in mammalian cells;enhancer/promoter sequences from the immediate early gene of human CMVfor high levels of transcription; transcription termination and RNAprocessing signals from SV40 for mRNA stability; SV40 polyoma origins ofreplication and ColE1 for proper episomal replication; versatilemultiple cloning sites; and T7 and SP6 RNA promoters for in vitrotranscription of sense and antisense RNA. Suitable vectors and methodsfor producing vectors containing transgenes are well known and availablein the art. Sambrook et al., Molecular Cloning, A Laboratory Mannual,3^(rd) Ed., Cold Spring Harbor Laboratory Press.

A “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. The vector can becapable of autonomous replication or integrate into a host DNA. Examplesof the vector include a plasmid, cosmid, or viral vector. The vectorsfor use in the methods described herein may include a nucleic acid in aform suitable for expression of the nucleic acid in a host cell.Preferably the vector includes one or more regulatory sequencesoperatively linked to the nucleic acid sequence to be expressed. Theterm “regulatory sequence” includes promoters, enhancers, and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those that direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof transcription of RNA desired, and the like.

Selection of a suitable vector may depend on the type of host cell, towhich the vector is to be introduced. For example, a bacterial vectormay be selected if it is to be introduced into a bacterial cell. In someexamples, a viral vector may be used for introducing nucleic acids thatencode a fusion polypeptide as described herein into an immune cell(e.g., a T cell, a B cell, a natural killer cell, a dendritic cell, or amacrophage). A “viral vector” as described herein refers to arecombinantly produced virus or viral particle that comprises apolynucleotide to be delivered into a host cell, either in vivo, ex vivoor in vitro. Examples of viral vectors include retroviral vectors suchas lentiviral vectors, adenovirus vectors, adeno-associated virusvectors and the like. In aspects where gene transfer is mediated by aretroviral vector, a vector construct refers to the polynucleotidecomprising the retroviral genome or part thereof, and a therapeuticgene.

A variety of promoters can be used for expression of a polypeptidedescribed herein, e.g., a polypeptide comprising a sortase/activefragment thereof or a sortase acceptor peptide, which may or may not befused to a member of a ligand-receptor pair. Promoters that can be usedto express the protein are well known in the art. Promoters includecytomegalovirus (CMV) intermediate early promoter, a viral LTR such asthe Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40(SV40) early promoter, E. coli lac UV5 promoter and the herpes simplextk virus promoter.

Regulatable promoters can also be used. Such regulatable promotersinclude those using the lac repressor from E. coli as a transcriptionmodulator to regulate transcription from lac operator-bearing mammaliancell promoters (Brown, M. et al., Cell, 49:603-612 (1987)), those usingthe tetracycline repressor (tetR) (Gossen, et al., Proc. Natl. Acad.Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy,9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA,92:6522-6526 (1995)). Other systems include FK506 dimer, VP16 or p65using estradiol, RU486, diphenol murislerone or rapamycin. Induciblesystems are available from Invitrogen, Clontech and Ariad.

Regulatable promoters that include a repressor with the operon can beused. In one embodiment, the lac repressor from E. coli can function asa transcriptional modulator to regulate transcription from lacoperator-bearing mammalian cell promoters (Brown et al., Cell,49:603-612 (1987)); Gossen and Bujard (1992); Gossen et al., Natl. Acad.Sci. USA, 89:5547-5551 (1992)) combined the tetracycline repressor(tetR) with the transcription activator (VP 16) to create atetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP16), with the tetO-bearing minimal promoter derived from the humancytomegalovirus (hCMV) major immediate-early promoter to create atetR-tet operator system to control gene expression in mammalian cells.In one embodiment, a tetracycline inducible switch is used. Thetetracycline repressor (tetR) alone, rather than the tetR-mammalian celltranscription factor fusion derivatives can function as potenttrans-modulator to regulate gene expression in mammalian cells when thetetracycline operator is properly positioned downstream for the TATAelement of the CMVIE promoter (Yao et al., Human Gene Therapy). Oneparticular advantage of this tetracycline inducible switch is that itdoes not require the use of a tetracycline repressor-mammalian cellstransactivator or repressor fusion protein, which in some instances canbe toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551(1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526(1995)), to achieve its regulatable effects.

The effectiveness of some inducible promoters can be increased overtime. In such cases one can enhance the effectiveness of such systems byinserting multiple repressors in tandem, e.g., TetR linked to a TetR byan internal ribosome entry site (IRES). Alternatively, one can wait atleast 3 days before screening for the desired function. While somesilencing may occur, it can be minimized by using a suitable number ofcells, preferably at least 1×10⁴, more preferably at least 1×10⁵, stillmore preferably at least 1×10⁶, and even more preferably at least 1×10⁷.One can enhance expression of desired proteins by known means to enhancethe effectiveness of this system. For example, using the WoodchuckHepatitis Virus Posttranscriptional Regulatory Element (WPRE). See Loeb,V. E., et al., Human Gene Therapy 10:2295-2305 (1999); Zufferey, R., etal., J. of Virol. 73:2886-2892 (1999); Donello, J. E., et al., J. ofVirol. 72:5085-5092 (1998).

Examples of polyadenylation signals useful to practice the methodsdescribed herein include, but are not limited to, human collagen Ipolyadenylation signal, human collagen II polyadenylation signal, andSV40 polyadenylation signal.

Vectors comprising nucleic acid sequences encoding the fusionpolypeptides described herein, which may be operably linked toregulatory elements, may remain present in the cell as a functioningcytoplasmic molecule, a functioning episomal molecule or it mayintegrate into the cell's chromosomal DNA. Exogenous genetic materialmay be introduced into cells where it remains as separate geneticmaterial in the form of a plasmid. Alternatively, linear DNA, which canintegrate into the chromosome, may be introduced into the cell. Whenintroducing DNA into the cell, reagents, which promote DNA integrationinto chromosomes, may be added. DNA sequences, which are useful topromote integration, may also be included in the DNA molecule.Alternatively, RNA may be introduced into the cell.

Selectable markers can be used to monitor uptake of the desiredtransgene into the progenitor cells described herein. These marker genescan be under the control of any promoter or an inducible promoter. Theseare known in the art and include genes that change the sensitivity of acell to a stimulus such as a nutrient, an antibiotic, etc. Genes includethose for neo, puro, tk, multiple drug resistance (MDR), etc. Othergenes express proteins that can readily be screened for such as greenfluorescent protein (GFP), blue fluorescent protein (BFP), luciferase,and LacZ.

Any of the engineered cells or a combination thereof are also within thescope of the present disclosure.

Any of the intercellular labeling methods described herein may be usedto track cell-cell interaction either in vitro or in vivo. Someexemplary utilities are provided below.

(iii) In Vitro Labeling

The intercellular labeling methods may be performed either in vitro orin vivo. In some embodiments, the intercellular labeling methods can beperformed in vitro, where the two engineered cells described herein areincubated in a cell culture in the presence of a peptide that comprisesa sortase recognition sequence (“sortase substrate”). Such a peptide isconjugated with a detectable label. When the ligand-receptor interact,the resultant spatial proximity would allow for the transfer of thedetectable label onto the sortase acceptor peptide via thetranspeptidation reaction catalyzed by the sortase, leading to thelabeling of the cell that expresses the sortase acceptor peptide.

The sortase substrate used in the methods described herein, which isconjugated to a detectable label, can comprise any sortase recognitionsequence as known in the art or disclosed herein. Selection of asuitable sortase recognition sequence would depend on the type ofsortase used in the same methods.

For example, when a sortase A is used, the corresponding substrate maycomprise the recognition sequence LPXTG (SEQ ID NO: 1), in which X canbe any amino acid residue (naturally-occurring or non-naturallyoccurring), e.g., any of the 20 standard amino acids found most commonlyin proteins found in living organisms. Alternatively, the recognitionmotif can be LPXT, in which X is D, E, A, N, Q, K, or R. In otherexamples, X is selected from K, E, N, Q, and A in an LPXTG (SEQ IDNO: 1) or LPXT motif, which are recognized by a sortase A. In someexamples, a mutant SrtA as described here is used and the correspondinglabeled substrate may comprise the motif LPETG (SEQ ID NO: 2). Suchmutants may comprise one or more of the following positions: P94, S102,A104, E105, K138, K152, D160, K162, T164, D165, K173, 1182, K190, andK196. For example, a SrtA mutant may comprise one or more of thefollowing mutations: P94R S102C, A104H, E105D, K138P, K152I, D160K,K162H, T164N, D165A, K173E, I182V, K190E, and K196S. In one example, thesortase is a triple mutant P94S/D160N/K196T of SrtA from S. aureus.

In an example, a sortase substrate comprising the recognition sequenceLPXTG (SEQ ID NO: 1) or LPXT, in which X is selected from K, S, E, L, A,and N, can be used when the corresponding sortase is a class C sortase.Exemplary sortase recognition motifs include, but are not limited to,LPKTG (SEQ ID NO: 8), LPITG (SEQ ID NO: 33), LPDTA (SEQ ID NO: 34),SPKTG (SEQ ID NO: 35), LAETG (SEQ ID NO: 20), LAATG (SEQ ID NO: 36),LAHTG (SEQ ID NO: 37), LASTG (SEQ ID NO: 38), LPLTG (SEQ ID NO: 39),LSRTG (SEQ ID NO: 40), LPETG (SEQ ID NO: 2), VPDTG (SEQ ID NO: 41),IPQTG (SEQ ID NO: 42), YPRRG (SEQ ID NO: 43), LPMTG (SEQ ID NO: 44),LAFTG (SEQ ID NO: 45), LPQTS (SEQ ID NO: 46), LPXT, LAXT, LPXA, LGXT,IPXT, NPXT, NPQS (SEQ ID NO: 47), LPST (SEQ ID NO: 48), NSKT (SEQ ID NO:49), NPQT (SEQ ID NO: 50), NAKT (SEQ ID NO: 51), LPIT (SEQ ID NO: 52),or LAET (SEQ ID NO: 53).

In some embodiments, a sortase substrate used in the labeling methods asdescribed herein can further comprises one or more additional aminoacids, e.g., at the N or C terminus of the sortase recognition sequence.For example, one or more amino acids (e.g., up to 5 amino acids) havingthe identity of amino acids found immediately N-terminal to, orC-terminal to, a five (5) amino acid recognition sequence in a naturallyoccurring sortase substrate may be incorporated. Such additional aminoacids may provide context that improves the recognition of therecognition motif. In some examples, a sortase substrate may contain upto 50 amino acid residues, for example, up to 40, 30, 20, 15, 10, or 5amino acid residues.

In some embodiments, the sortase recognition sequence in the sortasesubstrate can be masked. In contrast to an unmasked sortase recognitionmotif, which can be recognized by a sortase, a masked sortaserecognition motif is a motif that is not recognized by a sortase butthat can be readily modified (“unmasked”) such that the resulting motifis recognized by the sortase. For example, in some embodiments, at leastone amino acid of a masked sortase recognition motif comprises a sidechain comprising a moiety that inhibits, e.g., prevents, recognition ofthe sequence by a sortase of interest, e.g., SrtAaureus. Removal of theinhibiting moiety, in turn, allows recognition of the motif by thesortase. Masking may, for example, reduce recognition by at least 80%,90%, 95%, or more (e.g., to undetectable levels) in certain embodiments.By way of example, in certain embodiments a threonine residue in asortase recognition motif such as LPXTG (SEQ ID NO: 1) may bephosphorylated, thereby rendering it refractory to recognition andcleavage by SrtA. The masked recognition sequence can be unmasked bytreatment with a phosphatase, thus allowing it to be used in aSrtA-catalyzed transamidation reaction.

A sortase substrate used in the labeling methods described herein isconjugated to a detectable label. The term “conjugated” or “conjugation”refers to an association of two molecules, for example, two proteins ora protein and an agent, e.g., a small molecule, with one another in away that they are linked by a direct or indirect covalent ornon-covalent interaction. In certain embodiments, the association iscovalent, and the entities are said to be “conjugated” to one another.In some embodiments, a protein is post-translationally conjugated toanother molecule, for example, a second protein, a small molecule, adetectable label, a click chemistry handle, or a binding agent, byforming a covalent bond between the protein and the other molecule afterthe protein has been formed, and, in some embodiments, after the proteinhas been isolated. In some embodiments, two molecules are conjugated viaa linker connecting both molecules. For example, in some embodimentswhere two proteins are conjugated to each other to form a proteinfusion, the two proteins may be conjugated via a polypeptide linker,e.g., an amino acid sequence connecting the C-terminus of one protein tothe N-terminus of the other protein. In some embodiments, two proteinsare conjugated at their respective C-termini, generating a C—Cconjugated chimeric protein. In some embodiments, two proteins areconjugated at their respective N-termini, generating an N—N conjugatedchimeric protein. In some embodiments, conjugation of a protein to apeptide is achieved by transpeptidation using a sortase. See, e.g.,Ploegh et al., WO/2010/087994, and Ploegh et al., WO/2011/133704, andPloegh et al., International PCT Application PCT/US2014/037545, filedMay 9, 2014, the entire contents of each of which are incorporatedherein by reference, for exemplary sortases, proteins, recognitionmotifs, reagents, and methods for sortase-mediated transpeptidation.

A detectable label is a moiety that has at least one element, isotope,or functional group incorporated into the moiety which enables detectionof the molecule, e.g., a protein or peptide, or other entity, to whichthe label is attached. Labels can be directly attached (i.e., via abond) or can be attached by a linker (such as, for example, anoptionally substituted alkylene; an optionally substituted alkenylene;an optionally substituted alkynylene; an optionally substitutedheteroalkylene; an optionally substituted heteroalkenylene; anoptionally substituted heteroalkynylene; an optionally substitutedarylene; an optionally substituted heteroarylene; an optionallysubstituted acylene, or any combination thereof, which can make up alinker). It will be appreciated that the label may be attached to orincorporated into a molecule, for example, a protein, polypeptide, orother entity, at any position. In general, a detectable label can fallinto any one (or more) of five classes: a) a label which containsisotopic moieties, which may be radioactive or heavy isotopes,including, but not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P,³⁵S, ⁶⁷Ga, ⁷⁶Br, ⁹⁹mTc (Tc-99m), n, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁵³Gd, ¹⁶⁹Yb, and¹⁸⁶Re; b) a label which contains an immune moiety, which may beantibodies or antigens, which may be bound to enzymes (e.g., such ashorseradish peroxidase); c) a label which is a colored, luminescent,phosphorescent, or fluorescent moieties (e.g., such as the fluorescentlabel fluorescein-isothiocyanate (FITC); d) a label which has one ormore photo affinity moieties; and e) a label which is a ligand for oneor more known binding partners (e.g., biotin-streptavidin, FK506-FKBP).In certain embodiments, a label comprises a radioactive isotope,preferably an isotope which emits detectable particles, such as βparticles. In certain embodiments, the label comprises a fluorescentmoiety. In certain embodiments, the label is the fluorescent labelfluorescein-isothiocyanate (FITC). In certain embodiments, the labelcomprises a ligand moiety with one or more known binding partners. Incertain embodiments, the label comprises biotin. In some embodiments, alabel is a fluorescent polypeptide (e.g., GFP or a derivative thereofsuch as enhanced GFP (EGFP)) or a luciferase (e.g., a firefly, Renilla,or Gaussia luciferase). It will be appreciated that, in certainembodiments, a label may react with a suitable substrate (e.g., aluciferin) to generate a detectable signal. Non-limiting examples offluorescent proteins include GFP and derivatives thereof, proteinscomprising fluorophores that emit light of different colors such as red,yellow, and cyan fluorescent proteins. Exemplary fluorescent proteinsinclude, e.g., Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP,Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2, EGFP, mWasabi,EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mKO2, mOrange,mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry,mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima. See, e.g.,Chalfie, M. and Kain, S R (eds.) Green fluorescent protein: properties,applications, and protocols Methods of biochemical analysis, v. 47Wiley-Interscience, Hoboken, N.J., 2006; and Chudakov, D M, et al.,Physiol Rev. 90(3):1103-63, 2010, for discussion of GFP and numerousother fluorescent or luminescent proteins. In some embodiments, a labelcomprises a dark quencher, e.g., a substance that absorbs excitationenergy from a fluorophore and dissipates the energy as heat.

Labeling of a cell expressing a sortase acceptor peptide fused to amember of a ligand-receptor pair can be detected via a routine method,depending upon the detectable label conjugated to the sortase substrate.For example, if a fluorescent dye or a label that releases a detectablesignal is used, the labeled cells can be detected by, e.g., FACSanalysis. In another example, the sortase substrate is conjugated tobiotin and the cells can be further incubated with streptavidin and thenanalyzed by, e.g., FACS. When the fusion protein further comprises aprotein tag, a labeled antibody specific to the protein tag can be usedfor detected labelled cells.

Cells that are labelled in the labelling methods described herein can beisolated and subject to further analysis, as described herein.

(iv) In Vivo Labeling

In other embodiments, the intercellular labeling methods described heremay be performed in vivo, for, e.g., tracking cell-cell interactions invivo. Such an in vivo assay can be performed in a suitable subject, forexample, a non-human animal such as a non-human mammal (e.g., mouse,rat, rabbit, dog, cat, or monkey).

In some examples, the two engineered cells described herein, oneexpressing a sortase or an active fragment thereof on the surface andthe other expressing a surface polypeptide comprising a sortase acceptorpeptide at the N-terminus, can be prepared in vitro following routinetechnology or as described herein. Either or both of the sortase/activefragment and the sortase acceptor peptide may or may not be fused to amember of a receptor-ligand pair. Those engineered cells can then betransferred into a suitable subject via a suitable route, for example,intravenous infusion. The subject can be co-administered with a suitablelabelled sortase substrate as described herein.

In other examples, one or both of the nucleic acids encoding apolypeptide comprising the sortase/active fragment and the nucleic acidencoding a polypeptide comprising the sortase acceptor peptide can beintroduced into the genome of a suitable non-human animal (e.g.,zebrafish or a non-human mammal, such as a mouse, a rat, a rabbit, or amonkey) to make a transgenic animal. Alternatively, one or both of thenucleic acids encoding the sortase and/or the sortase acceptor peptidecan be inserted into the genome of a suitable non-human animal at theendogenous locus (loci) encoding one or both members of theligand-receptor pair for producing one or both the fusion polypeptidesin the transgenic animal. In this case, the endogenous gene encoding oneor both members of the ligand-receptor pair is used for producing thefusion polypeptides.

A “transgenic animal” as used herein can be any animal containing one ormore cells bearing genetic materials for expressing one or both of thefusion polypeptides described herein, directly or indirectly, bydeliberate genetic manipulation at the subcellular level, such as bytargeted recombination or microinjection or infection with recombinantvirus. The term “transgenic animal” is meant to encompass animals inwhich one or more cells receive a recombinant DNA molecule as describedherein. This molecule may be specifically targeted to a defined geneticlocus, be randomly integrated within a chromosome, or it may beextra-chromosomally replicating DNA. Any of the transgenic animals,either engineered to express one fusion polypeptide or both fusionpolypeptide as described herein, are within the scope of the presentdisclosure.

The transgenic animal described herein can be a non-human animal, e.g.,a mammal such as a rodent (e.g., a rat or mouse), in which an exogenousnucleic acid encoding a fusion polypeptide as described herein isinserted into its genome, i.e., one or more of the cells of the animalincludes a nucleic acid(s) encoding one or both of the fusionpolypeptides described herein. In some examples, it may have theexogenous nucleic acid sequence present as an extrachromosomal elementin a portion of its cell. In other examples, the exogenous nucleic acidscan be stably integrated into its germ line DNA (i.e., in the genomicsequence of most or all of its cells). Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, etc. Unless otherwise indicated, it will be assumed that atransgenic animal comprises stable changes to the germline sequence.Exogenous nucleic acid is introduced into the germ line of such atransgenic animal by genetic manipulation of, for example, embryos orembryonic stem cells of the host animal.

A transgene comprising an exogenous nucleic acid of interest for use inconstructing any of the transgenic animals described herein can beconstructed via routine technology. A transgene is a nucleotide sequencethat has been or is designed to be incorporated into a cell,particularly a mammalian cell, that in turn becomes or is incorporatedinto a living animal such that the nucleic acid containing thenucleotide sequence is expressed (i.e., the mammalian cell istransformed with the transgene). A transgene typically comprises thecoding sequence for an exogenous protein of interest (here a fusionpolypeptide as described herein) under the control of a regulatorysequences for a “characterizing gene.” The regulatory sequence can be anendogenous promoter of a characterizing gene. This characterizing geneis endogenous to a host cell or host organism (or is an ortholog of anendogenous gene) and is expressed in a particular select population ofcells of the organism (e.g., in immune cells such as T or B cells).

Methods for constructing transgenes are well known in the art,including, but not limited to, in vitro recombinant DNA techniques andin vivo genetic recombination. See, e.g., Sambrook et al., 2001,Molecular Cloning, A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y., both of which are hereby incorporated by referencein their entireties.

Introduction of a nucleic acid encoding an exogenous polypeptide (e.g.,a transgene as described herein) can be achieved by a variety of methodsincluding, for example, microinjection of half-day embryo pronuclei,transfection of embryonic stem cells, or fusion of embryonic stem cellswith yeast spheroplasts or micronuclei comprising transchromosomes. Thetransgenic animal such as transgenic mammals (non-human) resulting fromthe processes described herein are capable of functionally rearrangingthe introduced exogenous genetic sequences, and expressing one or bothof the fusion polypeptides as described herein. Transgenic nonhumanmammals such as rodents (e.g., mice) are particularly suitable for usein the methods described herein.

In some examples, the exogenous nucleic acids as described herein of mayintegrate into the genome of a suitable animal recipient (or an oocyteor embryo that gives rise to the recipient organism), e.g., by randomintegration or site-specific integratin. If random, the integrationpreferably does not knock out, e.g., insert into, an endogenous gene(s)such that the expression of the endogenous gene is not affected.Alternatively, the exogenous nucleic acid may integrate by a directedmethod, e.g., by directed homologous recombination (“knock-in”),Chappel, U.S. Pat. No. 5,272,071; and PCT publication No. WO 91/06667,published May 16, 1991; U.S. Pat. No. 5,464,764; Capecchi et al., issuedNov. 7, 1995; U.S. Pat. No. 5,627,059, Capecchi et al. issued, May 6,1997; U.S. Pat. No. 5,487,992, Capecchi et al., issued Jan. 30, 1996).Preferably, when homologous recombination is used, it does not knock outor replace the host's endogenous copy of the characterizing gene.

Methods for generating cells having targeted gene modifications throughhomologous recombination are known in the art. The construct willcomprise at least a portion of the characterizing gene with a desiredgenetic modification, e.g., insertion of the nucleotide sequence codingfor the tagged ribosomal protein and will include regions of homology tothe target locus, i.e., the endogenous copy of the characterizing genein the host's genome. DNA constructs for random integration need notinclude regions of homology to mediate recombination. Markers can beincluded for performing positive and negative selection for insertion ofthe nucleic acid of the invention.

To create a homologous recombinant transgenic animal, a homologousrecombination vector can be prepared, in which the exogenous nucleotidesequence encoding the fusion polypeptide is flanked at its 5′ and 3′ends by characterizing gene sequences to allow for homologousrecombination to occur between the exogenous gene carried by the vectorand the endogenous characterizing gene in an embryonic stem cell of theanimal. The additional flanking nucleic acid sequences are of sufficientlength for successful homologous recombination with the endogenouscharacterizing gene. Typically, several kilobases of flanking DNA (bothat the 5′ and 3′ ends) are included in the vector. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are known in the art. See, e.g., Thomas and Capecchi, 1987, Cell51: 503; Bradley, 1991, Curr. Opin. Bio/Technol. 2: 823-29; and PCTPublication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

Alternatively, a homologous recombination vector can be prepared, inwhich the exogenous nucleotide sequence encoding the sortase or thesortase acceptor peptide is flanked at its 5′ and 3′ ends by sequenceshomologous to the endogenous gene locus for a member of theligand-receptor pair to allow for homologous recombination to occurbetween the exogenous gene carried by the vector and the endogenous genelocus in an embryonic stem cell of the animal. The homologousrecombination is designed such that fusion polypeptide(s) comprising thesortase and one member of the ligand-receptor pair, the sortase acceptorpeptide and the other member of the ligand-receptor pair, or both can beproduced in the transgenic animal thus prepared. The additional flankingnucleic acid sequences are of sufficient length for successfulhomologous recombination with the endogenous characterizing gene.Nucleotide sequences encoding the sortase or the sortase acceptorpeptide can also be inserted into the endogenous gene locus for one orboth of the members of the ligand-receptor pair via site-specificrecombination, e.g., via the Cre/LoxP system known in the art.

In one example, a transgenic animal of the present disclosure can becreated by introducing one or more nucleic acids encoding one or both ofthe fusion polypeptide operably linked to suitable promoters into themale pronuclei of a fertilized oocyte, e.g., by microinjection orretroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, are well known in the art, see, e.g., U.S. Pat. Nos.4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191, Hogan, Manipulatingthe Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1986) and Wakayama et al., 1999, Proc. Natl. Acad. Sci.USA, 96:14984-89. Similar methods are used for production of othertransgenic animals.

In another embodiment, the exogenous nucleic acid as described hereincan be inserted into the genome of an embryonic stem (ES) cell or aninduced pluripotent stem cells (iPS) cell, followed by injection of themodified ES cell or iPS cell into a blastocyst-stage embryo thatsubsequently develops to maturity and serves as the founder animal for aline of transgenic animals.

For example, a vector comprising the exogenous nucleic acid can beintroduced into ES cells (e.g., by electroporation) and cells in whichthe introduced gene has homologously recombined with the endogenous geneare selected. See, e.g., Li et al., 1992, Cell 69:915. For embryonicstem (ES) cells, an ES cell line may be employed, or embryonic cells maybe obtained freshly from a host, e.g., mouse, rat, guinea pig, etc.

After transformation, ES cells can be grown on an appropriate feederlayer, e.g., a fibroblast-feeder layer, in an appropriate medium and inthe presence of appropriate growth factors, such as leukemia inhibitingfactory (LIF). Cells that contain the construct may be detected byemploying a selective medium. Transformed ES cells may then be used toproduce transgenic animals via embryo manipulation and blastocystinjection. (See, e.g., U.S. Pat. Nos. 5,387,742, 4,736,866 and 5,565,186for methods of making transgenic animals.)

Stable expression of the construct is preferred. For example, ES cellsthat stably express a nucleotide sequence encoding a fusion polypeptideas described herein may be engineered. ES host cells can be transformedwith DNA, e.g., a plasmid, controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the exogenous DNA, engineered ES cells maybe allowed to grow for 1-2 days in an enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and expanded into celllines.

The selected ES cells can then injected into a blastocyst of an animal(e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987,in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,Robertson, ed., IRL, Oxford, 113-52. Blastocysts can be obtained from 4to 6 week old superovulated females. The ES cells can be trypsinized,and the modified cells can be injected into the blastocoel of theblastocyst. After injection, the blastocysts can be implanted into theuterine horns of suitable pseudopregnant female foster animal.Alternatively, the ES cells may be incorporated into a morula to form amorula aggregate which is then implanted into a suitable pseudopregnantfemale foster animal. Females are then allowed to go to term and theresulting litters screened for mutant cells having the construct.

The chimeric animals can be screened for the presence of the modifiedgene. By providing for a different phenotype of the blastocyst and theES cells, chimeric progeny can be readily detected. Males and femalechimeras having the modification are mated to produce homozygousprogeny. Only chimeras with transformed germline cells would generatehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allergenicor congenic grafts or transplants, or in in vitro culture.

Progeny harboring homologously recombined or integrated exogenous DNA intheir germline cells can be used to breed animals in which all cells ofthe animal contain the homologously recombined DNA by germlinetransmission of the exogenous nucleic acid as described herein. Clonesof the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al., 1997,Nature 385: 810-13 and PCT Publication NOS. WO 97/07668 and WO 97/07669.Once the transgenic mice are generated, they may be bred and maintainedusing methods well known in the art. See, e.g., Manipulating the MouseEmbryo. A Laboratory Manual, 2nd edition. B. Hogan, Beddington, R.,Costantini, F. and Lacy, E., eds. 1994. Cold Spring Harbor LaboratoryPress: Plainview, N.Y.

A transgenic founder animal can be identified based upon the presence ofthe exogenous nucleic acid in its genome and/or expression of mRNAencoding the fusion polypeptide described herein in tissues or cells ofthe animals. A transgenic founder animal can then be used to breedadditional animals carrying the exogenous nucleic acid as describedherein. Moreover, transgenic animals carrying the exogenous nucleic acidcan further be bred to other transgenic animals carrying other exogenousnucleic acids. For example, a transgenic animal carrying the exogenousnucleic acid encoding the fusion polypeptide comprising a sortase andone member of a ligand-receptor pair can be bred with another transgenicanimal carrying the exogenous nucleic acid encoding the fusionpolypeptide comprising a sortase acceptor peptide and the other memberof the ligand-receptor pair to produce a double knock-in transgenicanimal.

In some embodiments, genome editing technology (also known as genomeediting with engineered nucleases or GEEN) may be used to modifyendogenous genes of interest for making the transgenic animals asdescribed herein. Gene editing is a type of genetic engineering, inwhich DNA is inserted, replaced, or removed from a genome usingartificially engineered nucleases, e.g., Zinc finger nucleases (ZFNs),Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cassystem, and engineered meganuclease re-engineered homing endonucleases.See, e.g., Esvelt, et al., (2013), Mol Syst Biol 9 (1): 641; Tan et al.,(2012), Adv Genet 80: 37-97; and Puchta et al., (2013), Int. J. Dev.Biol 57: 629-637. Any of such genome editing technology can be used forpreparing the transgenic animals described herein.

In other embodiments, a transgenic animal such as transgenic mousecapable of expressing one fusion polypeptide as described herein can beconstructed following the methods described herein. Such a transgenicanimal can then be administered with engineered cells expressing theother fusion polypeptide via routine methods. For example, a transgenicanimal capable of expressing a fusion polypeptide comprising a sortaseand one member of a ligand-receptor pair can be administered withengineered cells expressing a fusion polypeptide comprising a sortaseacceptor peptide fused to the other member of the ligand-receptor pair,or vice versa.

An animal or subject that express both fusion polypeptides as describedherein can then be administered with a labeled sortase substrate asdescribed herein. In some examples, the labeled sortase substrate andengineered cells expressing one or both fusion polypeptides areco-administered into a subject or transgenic animal.

The term “co-administration” is meant to refer to a combinedadministration by a suitable route(s), in which two or more agents areadministered to a subject. The agents may be in the same dosageformulations or separate formulations. For combined administration withmore than one agent, where the agents are in separate dosageformulations, the agents can be administered concurrently, or they eachcan be administered at separately staggered times. The agents may beadministered simultaneously or sequentially (e.g., one agent maydirectly follow administration of the other or the agents may be giveepisodically, e.g., one can be given at one time followed by the otherat a later time, e.g., within a week), as long as they are given in amanner sufficient to allow both agents to achieve effectiveconcentrations in the body. The agents may also be administered bydifferent routes, e.g., one agent may be administered intravenouslywhile a second agent is administered intramuscularly or orally. Thus,the engineered cells may be administered prior to, concomitant with, orafter the administration of the labelled sortase substrate.Co-administrable agents also may be formulated as an admixture, as, forexample, in a single formulation. These formulations may be parenteralor oral, such as the formulations described, e.g., in U.S. Pat. Nos.6,277,384; 6,261,599; 5,958,452 and PCT publication No. WO 98/25613,each hereby incorporated by reference.

Afterwards, cells expressing the fusion polypeptide comprising thesortase acceptor peptide and one member of the ligand-receptor pair canbe either detected in vivo, e.g., by imaging, or isolated from thesubject and subjected to further analysis in vitro (e.g., by FACSanalysis).

II. Kits

Some aspects of the present disclosure provide kits useful forperforming the intercellular labeling methods described herein, which iscatalyzed by a sortase and mediated by ligand-receptor interaction. Sucha kit can comprise (a) a first cell expressing a first polypeptide thatcomprise a sortase acceptor peptide as described herein, which islocated at the N-terminus of the first polypeptide, and (b) a secondcell expressing a second polypeptide that comprise a suitable sortase oran active fragment thereof. In some embodiments, the first polypeptidefurther comprises a member of a receptor-ligand pair, such as thosedescribed herein, and the second polypeptide further comprises the othermember of the receptor-ligand pair. In other embodiments, the firstpolypeptide and one member of the receptor-ligand pair are expressed onthe first cell as separate polypeptides. The second polypeptide and theother member of the receptor-ligand pair can also be expressed on thesurface of the second cell as separate polypeptide.

The kit may further comprise any of the labeled sortase substratedescribed herein. The sortase can transfer a suitable sortase substrateas described herein onto the acceptor peptide. In some embodiments sucha kit may comprise one or more nucleic acids that encode the first orsecond polypeptide, e.g., nucleic acids that can be inserted into avector or used directly to generate cells that express the first orsecond polypeptide.

In some embodiments, the kit may comprise (i) a plurality ofantigen-presenting cells (APCs), such as B cells, macrophages, or DCs;(ii) a T cell; and optionally (iii) a labeled sortase substrate. TheAPCs express one or both of MHC Class I and MHC Class II complexes.Further, the APC s express a polypeptide comprising a sortase acceptorpeptide, which is located at the N-terminus of the polypeptide.Collectively, the plurality of APCs also express polypeptides encoded bya cDNA library. The T cell in the kit expresses a T cell receptor, whichmay be endogenous or exogenous. Such a T cell also express a polypeptidecomprising a sortase such as SrtA or an active fragment thereof. Such akit can be used to identify the cognate antigen of a T cell receptorwith unknown specificity.

Alternatively or in addition, the kits described herein can comprise oneor more of the medium components for use in in vitro culturing of thecells described herein, e.g., one or more growth factors and nutritionalfactors for cell growth. If immune cells are involved, the mediumcomponents might include suitable cytokines needed for maintenance andgrowth of the immune cells.

Further, the kit can comprise a suitable sortase substrate which isconjugated to a detectable label as described herein. The selection ofthe suitable sortase substrate would depend on the sortase used in thekit. See above discussions.

In some embodiments, the kit further comprises a buffer or reagentuseful for carrying out a sortase-mediated transpeptidation reaction.

III. Application of the Intercellular Labelling Methods

The intercellular labeling method described herein provides a solutionfor tracking molecule interactions, particularly in vivo, which has notbeen achieved in the art. This method may be used for various purposes,including, but not limited to, identifying agents capable of modulatingthe interaction between a specific receptor and its cognate ligand,identifying B cells expressing high affinity B-cell receptors (thus highaffinity antibodies) by tracking interaction between B cells andfollicular T helper cells in germinal centers, or identifying a bindingpartner of a protein of interest.

(i) Screening for Agents Capable of Modulating Ligand-ReceptorInteraction

To screen for potential modulators of a ligand-receptor pair (e.g.,inhibitors or enhancers), a candidate agent can be co-cultured with thepair of cells described herein, one expressing a polypeptide whichcomprises a sortase or an active fragment thereof and one member of areceptor-ligand pair, and the other cell expressing a polypeptidecomprising a corresponding sortase acceptor peptide and the other memberof the ligand-receptor pair in the presence of a suitable labeledsortase substrate, which comprises a sequence that is recognizable bythe just-noted sortase. In some instances, the polypeptide comprising asortase or the active fragment thereof and the member of areceptor-ligand pair are expressed as separate polypeptides on the cellsurface. In other instances, they are expressed as one fusionpolypeptide. Alternatively or in addition, the sortase acceptor peptideand the other member of the receptor-ligand pair may be expressed asseparate polypeptides on cell surface or expressed as one fusionpolypeptide.

The labeling level of the cell expressing the fusion polypeptidecomprising the sortase acceptor peptide is measured in the presence andabsence of the candidate agent. If the level of labeling changes in thepresence of the candidate agent as relative to in the absence of thecandidate agent, it indicates that the candidate agent is a potentialmodulator of the involved ligand-receptor pair. For example, if thelevel of labeling increases in the presence of the candidate agent, suchan agent might enhance the interaction between the ligand and receptor.On the other hand, if the level of labeling decreases in the presence ofthe candidate agent, such an agent might inhibit the interaction betweenthe ligand and receptor.

(ii) Identifying B Cells Expressing High Affinity B Cell Receptors

One of the key characteristics of the immune system is the ability toreact to pathogens by generating soluble molecules called antibodies orimmunoglobulins (Igs). An effective immune response requires theproduction of antibodies that bind antigen with high affinity andspecificity.

The immune system can generate antibodies capable of recognizing avirtually unlimited number of antigenic determinants. This requiresenormous variability among Igs. The first process accounting for thisvariability is VDJ recombination. VDJ recombination takes place in thebone marrow before antigen exposure. There, B cells undergocombinatorial rearrangement of V, D and J gene segments, which encodefor the antigen-binding portion of the Ig molecule. This mechanismprovides a first source of variability which, although broad, is stilllimited when compared to the vast number of antigens potentiallyrecognized by antibodies. Therefore, the first antibodies producedagainst an immunizing antigen are usually of low affinity.

Exposure to antigen triggers the generation and clonal selection of Bcells carrying novel mutant Ig sequences with improved antigen affinity,in a phenomenon known as affinity maturation. Affinity maturation is theresult of the combination of two processes: somatic hypermutation andaffinity-based selection, both of which occur in anatomic structuresreferred to as germinal centers (GCs). These structures arise insecondary lymphoid organs, such as the spleen and lymph nodes, about oneweek after primary antigen exposure. GCs are composed of twofunctionally distinct compartments: the light zone (LZ) and the darkzone (DZ). B cell proliferation is restricted to the DZ. Also in the DZ,insertion of somatic genetic mutations into Ig variable regions ensuresfurther expansion of Ig variability. Affinity-based selection of Igsafter somatic hypermutation occurs in the LZ. Recent studies pinpointedthe LZ decision-making process to the antigen-dependent interactionbetween GC B cells and follicular T helper (Tfh) cells, which are thelimiting factor in affinity-based selection of GC B cells. According tothe proposed model, B cells that exhibit high affinity Ig molecules atthe plasma membrane will capture and process more antigen forpresentation on Major Histocompatibility Complex (MHC) class IImolecules. A limiting number of Tfh cells then selects those B cellswith the highest peptide-MHC density and directs their return to the DZ,where they undergo rapid division. By contrast, B cells that fail tointeract with Tfh cells undergo apoptosis.

Despite the crucial role of the interaction between Tfh cells and Bcells in affinity maturation, little is known about how theseinteractions lead to selection of some cells and elimination of othersin vivo. This gap is due largely to the fact that there is no effectiveway to determine the extent to which two cells have interacted within aliving animal.

The intercellular labeling approach described herein would allow formeasuring interactions between immune cells in vivo during a GCreaction. Such a system could be used to distinguish between B cellsbased on the intensity of their interactions with other Tfh cells, andwould therefore provide a powerful tool to investigate Tfh-mediatedselection of GC B cells in a physiological setting and to identify Bcells expressing high affinity BCRs to antigens (thus high affinityantibodies). The labeling intensity is expected to correlate with BCRaffinity. The method can also be used to determine the ligand-receptorinteraction(s) that is important in in vivo selection of B cellsexpressing high affinity BCRs.

As used herein, a B cell receptor or BCR refers to a transmembranereceptor protein located on the outer surface of B-cells, comprising amembrane-bound antibody that, like all antibodies, has a unique andrandomly determined antigen-binding site. A BCR is composed of twoparts: (i) a membrane-bound immunoglobulin molecule of one isotype (IgG,IgD, IgM, IgA or IgE); and (ii) a signal transduction moiety, which isan Igα/Igβ heterodimer (CD79). Each member of the dimer spans the plasmamembrane and has a cytoplasmic tail bearing an immunoreceptortyrosine-based activation motif (ITAM).

As used herein, “binding affinity” refers to the apparent associationconstant or KA. The KA is the reciprocal of the dissociation constant(KD). The BCRs or antibodies isolated from the methods described hereinmay have a binding affinity (KD) of at least 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻,10⁻⁹, 10⁻¹⁰ M, or lower. An increased binding affinity corresponds to adecreased KD. Binding affinity can be determined by a variety of methodsincluding equilibrium dialysis, equilibrium binding, gel filtration,ELISA, surface plasmon resonance, or spectroscopy (e.g., using afluorescence assay). Exemplary conditions for evaluating bindingaffinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005%(v/v) Surfactant P20). These techniques can be used to measure theconcentration of bound binding antigen as a function of target antigenconcentration. The concentration of bound binding antigen ([Bound]) isrelated to the concentration of free target antigen ([Free]) and theconcentration of binding sites for the binding antigen on the targetwhere (N) is the number of binding sites per target molecule by thefollowing equation:

[Bound]=[N][Free]/(Kd+[Free])

It is not always necessary to make an exact determination of K_(A),though, since sometimes it is sufficient to obtain a quantitativemeasurement of affinity, e.g., determined using a method such as ELISAor FACS analysis, is proportional to K_(A), and thus can be used forcomparisons, such as determining whether a higher affinity is, e.g.,2-fold higher, to obtain a qualitative measurement of affinity, or toobtain an inference of affinity, e.g., by activity in a functionalassay, e.g., an in vitro or in vivo assay.

To perform the method for identifying B cells expressing high affinityBCRs from, e.g., a germinal center, a non-human mammal (e.g., mouse,rat, rabbit, or monkey) may be provided, which carries (i) B cells(e.g., naive B cells) expressing a B cell receptor and surfacepolypeptide comprising a sortase acceptor peptide located at theN-terminus of the polypeptide, and (ii) a different type of immune cellssuch as T cells that express a surface polypeptide comprising a sortase.The immune cells such as T cells may also display a ligand that isrecognizable by the BCR. In some embodiments the surface polypeptidecomprising a sortase acceptor peptide and the surface polypeptidecomprising a sortase can be fused to members of a ligand-receptor pair(e.g., any of the ligand-receptor pairs described herein.

The non-human mammal can be administered with a labeled sortasesubstrate, which comprises a sortase recognition sequence. Any of thesortase, sortase acceptor peptides, and sortase substrates that comprisesortase recognition sequences described herein can be used in thismethod. Selection of a suitable combination of the sortase, sortaseacceptor peptide, and sortase substrate is also described herein. Thelabeled sortase substrate delivered into the mammal would bind to theimmune cells such as T cells that express a surface fusion polypeptidecomprising a sortase. Upon interaction between the B cell and the immunecell (e.g., T cell) via BCR/ligand binding, the sortase catalyzes atranspeptidation reaction to transfer the labelled sortase substrateonto the sortase acceptor peptide on B cells. Lymphocytes can then beisolated from a suitable tissue, such as a germinal center or lymph nodeand labeled B cells can be purified for further analysis. Such labeled Bcells express high affinity BCRs (and thus high affinity antibodies) toantigens.

In some embodiments, the non-human mammal may be immunized with anantigen of interest following routine practice. Exemplary antigensinclude, but are not limited to, proteins or other biological moleculesexpressed by a pathogen (e.g., a bacterial pathogen or a viral pathogen)or the pathogen itself, optionally inactivated or attenuated, tumorantigens or tumor cells or oncogenic proteins. The resulting antibodymay in some embodiments be used as a therapeutic agent, e.g., to targeta pathogen or tumor.

Immunization of a host animal with a target antigen or a fragmentcontaining the target antigen conjugated to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using, for example, a bifunctional or derivatizing agent, forexample maleimidobenzoyl sulfosuccinimide ester (conjugation throughcysteine residues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl, or R¹N═C═NR, where R and R¹are different alkyl groups), can yield a population of antibodies (e.g.,polyclonal antibodies). Labeled B cells isolated from such a non-humanmammal may be subjected to further screening to identify those thatexpress high affinity BCRs specific to the antigen of interest.

The isolated labeled B cells can be cultured in vitro following routinemethods. Nucleic acids encoding the heavy chain, the light chain, orboth, or a portion thereof (e.g., a fragment comprising at least onecomplementarity determining region such as the variable regions of heavyand light chains) of the BCR can be isolated from the labeled B cells.The nucleic acids may be subjected to further sequencing to determinethe coding sequences. In some examples, a nucleic acid(s) encoding atleast the CDR3 of the heavy chain, the CDR3 of the light chain, or bothcan be isolated and sequenced. In other examples, a nucleic acid(s)encoding all of the CDR1-CDR3 of the heavy chain, all of the CDR1-CDR3of the light chain, or both can be isolated and sequenced. In yet otherexamples, a nucleic acid(s) encoding the whole variable region of theheavy chain, the whole variable region of the light chain, or both areisolated and sequenced. If the CDRs are derived from a non-human mammal,such CDRs can be grafted into a suitable human heavy chain or lightchain framework to produce humanized antibodies. Alternatively or inaddition, specificity determining residues (SDRs) may be identified,which may be grafted into a human heavy chain and/or light chainframework. Methods for isolating nucleic acids encoding a BCR heavyand/or light chain gene, or a portion thereof as described herein arewell known in the art. See, e.g., Mockridge et al., Clinical andExperimental Immunology, 114:129-136 (1998).

The nucleic acids thus isolated can be cloned into a suitable expressionvector for producing high affinity antibodies. The expression vector maybe introduced into a host cell, e.g., a mammalian host cell, such as aChinese hamster ovary (CHO) cell, an NSO murine myeloma cell, a PER.C6®human cell, etc., which may be used to produce the antibodies.

Alternatively, the isolated labeled B cells can be used to producehybridoma cell lines using the conventional hybridoma technology. Forexample, hybridomas can be prepared from the labeled B cells andimmortalized myeloma cells using the general somatic cell hybridizationtechnique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or asmodified by Buck, D. W., et al., In Vitro, 18:377-381 (1982). Availablemyeloma lines, including but not limited to X63-Ag8.653 and those fromthe Salk Institute, Cell Distribution Center, San Diego, Calif., USA,may be used in the hybridization. Generally, the technique involvesfusing myeloma cells and lymphoid cells using a fusogen such aspolyethylene glycol, or by electrical means well known to those skilledin the art. After the fusion, the cells are separated from the fusionmedium and grown in a selective growth medium, such ashypoxanthine-aminopterin-thymidine (HAT) medium, to eliminateunhybridized parent cells. Any of the media described herein,supplemented with or without serum, can be used for culturing hybridomasthat secrete monoclonal antibodies. As another alternative to the cellfusion technique, EBV immortalized B cells may be used to producemonoclonal high affinity antibodies, e.g., antibodies specific to theprotein of interest. The hybridomas can be expanded and subcloned, ifdesired, and supernatants are assayed for anti-immunogen activity byconventional immunoassay procedures (e.g., radioimmunoassay, enzymeimmunoassay, or fluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal high affinity antibodies. Hybridomas that produce suchantibodies may be grown in vitro or in vivo using known procedures. Themonoclonal antibodies may be isolated from the culture media or bodyfluids, by conventional immunoglobulin purification procedures such asammonium sulfate precipitation, gel electrophoresis, dialysis,chromatography, and ultrafiltration, if desired. Undesired activity ifpresent, can be removed, for example, by running the preparation overadsorbents made of the immunogen attached to a solid phase and elutingor releasing the desired antibodies off the immunogen.

The non-human mammal as described herein may be established bytransferring the B cells and a different type of immune cells (e.g., Tcells), which can be constructed in vitro, into a host non-human mammalvia a suitable route (e.g., intravenous infusion) as described herein.Alternatively, the non-human mammal may be a transgenic mammalengineered for producing (a) B cells expressing a surface polypeptidecomprising a sortase acceptor peptide and a B cell receptor, (b) adifferent type of immune cells such as T cells that express a surfacepolypeptide comprising a sortase or an active fragment thereof, or both(a) and (b). In one example, the non-human mammal is a double knock-intransgenic mammal that produces both (a) and (b). In another example,the non-human mammal is a single knock-in transgenic mammal thatproduces one of (a) and (b) and is transferred with the other via, e.g.,infusion. Any of the transgenic mammals described herein can be preparedby a method known in the art and/or those described herein. See abovedescriptions.

In some embodiments, the non-human mammal is a humanized mammal such asa humanized mouse or humanized rat engineered for producing fully humanantibodies or a fragment thereof such as human-mouse or human-ratchimeric antibodies. Such a humanized mammal may have one or more humanimmunoglobulin loci, or a portion thereof, inserted into thecorresponding mouse or rat endogenous immunoglobulin loci.Alternatively, such a humanized mammal may carry one of more humanimmunoglobulin genes, which may be inserted into one chromosome themammal or remain as extrachromosomal genetic material. Humanized miceexpressing human antibodies are known in the art, including theVelocImmune mice provided by Regeneron Pharmaceuticals, Inc., Kymouse™mice provided by Kymab, XenoMouse™ provided by Abgenix, and HuMAb miceprovided by Medarex/GenPharm). Methods for preparing transgenic micecapable of producing human antibodies are known in the art. See, e.g.,U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, and6,150,584, the relevant disclosures thereof are incorporated byreference herein. Alternatively, such a humanized mammal may benaturally deficient in its own immune system or have had its own immunesystem ablated and have reconstituted with the human immune system orhuman hematopoietic stem cells to produce human immune cells. Examplesinclude SCID mice, NOG mice, or NSG mice.

In some embodiments the intercellular labeling approach described hereinmay be used for measuring interactions between immune cells in vitro,e.g., for purposes of identifying B cells that express a BCR with highaffinity for an antigen of interest. For example, a population of Bcells expressing different BCRs (e.g., a library of B cells engineeredto express different BCRs or isolated from a subject) and a sortaseacceptor peptide such as G5 at the cell surface may be mixed with apopulation of CD4⁺ T cells and a soluble antigen of interest. The CD4⁺ Tcells express a sortase or an active fragment thereof at their cellsurface. The sortase may be expressed as part of a fusion protein with acell surface receptor or ligand (which may be one that is upregulatedupon a cognate interaction) or may be otherwise fused to a transmembranedomain or membrane anchoring domain that tethers the sortase to the cellmembrane and allows it to be exposed to the external environment. Bcells that express a BCR with high affinity for the antigen will take upmore of the antigen than B cells that express a lower affinity BCR andwill present it more highly (e.g., in the form of peptides bound to cellsurface MHC), causing interaction with CD4⁺ T cells that express a TCRthat is specific for the antigen. The mixture of B cells and CD4+ Tcells is contacted with a labeled sortase substrate, and the B cellsthat interact with the CD4⁺ T cells become labeled. Such B cells canthen be isolated (e.g., by FACS) and nucleic acid (e.g., DNA) encodingthe BCR may be isolated, thereby identifying the BCR, as describedabove.

(iii) Identifying Binding Partners of a Protein of Interest

The intercellular labeling methods described can also be used foridentifying binding partners of a protein of interest. For example, oneor more nucleotide sequences coding for a polypeptide comprising asortase as described herein and a protein of interest (e.g., a cellsurface receptor such as a receptor of an immune cell) can be introducedinto a first cell. The nucleotide sequences encoding the polypeptidecomprising the sortase and the protein of interest may be linked forproducing a fusion polypeptide or separated for expressing individualpolypeptides. Further, a plurality of genes encoding a plurality ofpolypeptide binding candidates, and a nucleotide sequence encoding apolypeptide comprising a sortase acceptor peptide at the N-terminus canbe introduced into a population of cells. The sortase acceptor peptidemay or may not fused to the polypeptide binding candidates. Theplurality of polypeptide binding candidates encompass potential bindingpartners for the protein of interest. When needed, each of the pluralityof polypeptides may further comprise a protein tag, which may facilitateprotein detection and purification.

The first cell and the population of cells can be incubated in thepresence of a suitable sortase substrate, which is associated with adetectable label under conditions allowing for occurrence of thetranspeptidation reaction catalyzed by the sortase to conjugate thelabeled sortase substrate to the sortase acceptor peptide. Cellsconjugated to the detectable label can then be isolated via a routinemethod, e.g., by cell sorting. The labeled cells thus identified can befurther analyzed to determine the polypeptide expressed on those cells,which can be are binding partners for the protein of interest. Thebinding activities of the thus identified polypeptides can be confirmedby a conventional binding assay, e.g., ELISA assay.

Any of the sortases, sortase substrates (which comprise a sortaserecognition sequence), and sortase acceptor peptides described hereincan be in this screening method. Selection of suitable combinations ofthe three components are also disclosed herein or known to those skilledin the art.

(iv) Identifying Antigen Specificity of T Cells

The intercellular labeling methods described herein may also be usefulto identify the cognate antigen of a T cell with unknown specificity. Anintercellular labeling system comprising the following components may beused to achieve this aim: (a) a plurality of antigen presenting cellsengineered to express a surface polypeptide that comprises an N-terminalsortase acceptor peptide (e.g., those described herein such as anoligoglycine fragment, e.g., G5 fragment; SEQ ID NO: 3); (b) a T cellengineered to express a surface polypeptide comprising a sortase or anactive fragment thereof such as those described herein (e.g., SrtA); and(c) a substrate of the sortase that contains a sortase recognitionsequence and is conjugated to a label, e.g., biotin, a fluorophore, orother detectable label such as those described herein.

In some instances, the sortase, the sortase acceptor peptide, or bothcan be fused with a cell surface protein that is upregulated upon acognate interaction. In some embodiments, the sortase expressed by the Tcell and the sortase acceptor peptide expressed by the APC s are notfused to members of the same ligand-receptor pair. For example, thesortase and the sortase acceptor peptide may be fused to receptors orligands that are not members of the same receptor-ligand pair (e.g.,PDGFR/CD40, PDGFR/CD86) or fused to any polypeptides that comprise atransmembrane domain or membrane anchor domain such that the sortase andthe sortase acceptor peptide are expressed at the cell surface andexposed to the extracellular environment. In some embodiments, either orboth the sortase and the sortase acceptor peptide are fused to atransmembrane domain of a receptor or ligand that is/are upregulatedupon a cognate interaction. In some embodiments the sortase or thesortase acceptor peptide, but not both, is fused to a transmembranedomain of a receptor or ligand that is upregulated upon cognateinteraction.

The plurality of APCs can be any type of cells capable of presentingantigens to T cells. Examples include, but are not limited to, B cellsDCs, or macrophages. Each of the APCs expresses a MHC Class I molecule,a MHC Class II molecule, or both, which may be of any suitable origin(e.g., human, murine or any other species of interest). In one example,the MHC Class I and/or MHC Class II is endogenous. In another example,one or more exogenous genes encoding the MHC Class I and/or MHC Class IImolecules are introduced into host APCs to produce the APCs for use inthe method described herein. When the T cell used in this method isobtained from a subject, at least one of the MHC Class I and/or MHCClass II molecules expressed on the APCs may match one HLA allele ofthat subject.

The plurality of the APCs described herein also express, collectively,polypeptides encoded by a cDNA library. The cDNA library can beconstructed from any source of interest, e.g., human, murine, or anyother species of interest.

In some embodiments the cDNA library may be constructed from cancercells or cancer tissue. In some embodiments, cancer cells or tissue maybe obtained from a biopsy or surgery or isolated from a blood sample.The cancer cells or cancer may be of any type (e.g., any of the varioustypes mentioned herein). Cancer cells may overexpress certain proteinsthat might be absent or expressed only at low levels in normal cellsand/or may express mutant proteins that might not be present in normalcells. Such proteins may, for example, serve as cancer antigens. In someembodiments it is of interest to identify those antigens, e.g.,particular antigens that are recognized by T cells and/or B cells andmay not be expressed by normal cells or may be expressed at a much lowerlevel by normal cells. In some embodiments, such identification may beuseful in the context of personalized medicine, e.g., generatingvaccines or cell therapies tailored for a particular cancer patient.

In some embodiments the cDNA library may be constructed from cells ortissues of a particular type, e.g., cells or tissues that are subject toimmune-mediated attack in an autoimmune disease. This may be useful,e.g., to identify particular antigens against which the immune systemmounts an attack in autoimmune disease.

The T cell used in the method described herein expresses a T cellreceptor (TCR) whose antigen specificity needs to be determined. In oneexample, the TCR is endogenous. In another example, one or moreexogenous genes encoding a TCR complex can be introduced into a host Tcell, which may have the endogenous TCR gene deleted or inactivated. TheT cell may be derived from a cancer patient, e.g., from a site wheretumor grows. Such a T cell may target a cancer antigen. Alternatively,the T cell may be derived from a site where infection occurs. Such a Tcell may target an antigen of a pathogen, e.g., a viral antigen or abacterial antigen. In other instances, the T cell may be derived from asite affected by an autoimmune disease, e.g., synovial fluid, pancreaticlymph node, or cerebral spinal fluid. Such T cells may be autoreactive Tcells targeting autoantigens.

To perform the method described herein, the plurality of APCs may bebrought into contact with the T cell in the presence of the labeledsortase substrate. The contacting step may be performed in vitro by,e.g., co-culturing the APC cells and the T cell in the presence of alabeled sortase substrate as described herein. Alternatively, thecontacting step may be performed in vivo, for example, the APC cells,the T cell, and the labeled sortase substrate can be delivered into asuitable host (e.g., mouse or rat) via a suitable route (e.g.,intravenous or subcutaneous injection). If an APC cell displays apeptide/MHC complex that can interact with the TCR on the T cell, thespatial proximity would allow the sortase expressed on the T cell totransfer the labeled sortase substrate onto the sortase acceptor peptideon the APC cell, thereby labeling the APC cell.

In some embodiments the T cell may be of a particular T cell subset,e.g., cytotoxic T cells (e.g., CD8⁺ T cells), helper T cells (e.g., CD4⁺T cells), or a subset thereof.

The labeled APCs may be recovered by a suitable method such as FACSsorting. When needed, the population of labeled APCs may be enrichedbefore sorting by, e.g., immune-magnetic isolation, using specificreagents recognizing the label attached to the substrate (e.g., biotin,or fluorophores). Such reagents may be commercially available. Aftersorting, the cDNA sequence carried by the labeled APC can be recoveredvia conventional technology, e.g., PCR amplification followed bysequencing analysis. The results obtained from the analysis would revealthe antigen specificity of the TCR of interest, which is encoded by amember of the cDNA library expressed in the T cell.

Once identified, the antigen could be produced (e.g., using standardprotein expression methods). The antigen, optionally combined with anadjuvant, may be administered to a subject in need thereof (e.g., as aprophylactic or therapeutic vaccine).

In some embodiments APCs, e.g., DCs, are contacted with the antigen exvivo (allowing them to take up and process the antigen) or areengineered to express the antigen, and then administered to a patient.The APCs may then interact with T cells in the patient to stimulate theimmune response. In some embodiments APCs, e.g., DCs, are contacted withthe antigen ex vivo (allowing them to take up and process the antigen)or are engineered to express the antigen and then contacted with T cellsex vivo. The T cells are subsequently administered to the patient). Insome embodiments, the T cells are autologous. In some embodiments, the Tcells may be expanded ex vivo prior to contacting them with APCs orafterwards.

Another application of the technology for identifying antigens that arerecognized by T cells may be in the context of immune responses againsttransplanted organs, cells, or tissues. One could isolate T cells thathave infiltrated into organs or tissues that may be subject toautoimmune attack or rejection and identify the antigen(s) recognized bysuch T cells.

In some embodiments, in the case of antigens that are recognized by Tcells in subjects suffering from an autoimmune disease or rejection oftransplanted organ, tissue, or cells, the identified antigen may be usedto induce tolerance to the antigen or used in other strategies to reduceor prevent an immune response against the antigen and thereby reduce anautoimmune response or immune response against a transplanted organ,tissue, or cells. Such strategies might include administeringantibodies, peptides or small molecules that block interaction betweenthe TCR and the antigen or specifically deplete T cells that recognizethe antigen.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES Example 1: Intercellular Labeling Mediated by CD40/CD40LInteraction

To track intercellular interactions between ligand and receptormolecules expressed at the plasma membrane of cells, ligand and receptorpairs were engineered by genetic fusion so that one of the interactingpartner expressed S. aureus SortaseA (SrtA) enzyme at its extracellularportion and the other one presented 5 extracellular N-terminal glycineresidues (FIG. 1A). A triple mutated version of SrtA enzyme(P94S-D160N-K196T) with improved catalytic properties as described byChen et al. PNAS, 2011 (FIG. 1B) was employed. Upon ligand-receptorinteraction, and in presence of a biotinylatated or fluorescentlylabeled SrtA substrate as the short peptide LPETG (SEQ ID NO: 2),spatial proximity allows the labeled substrate to be transferred fromthe SrtA-engineered ligand to the N-terminus glycine of the receptor.

Briefly, expression vectors carrying G₅-myc-CD40 and CD40L-SrtA fusionproteins as shown in FIG. 2A were constructed via routine recombinanttechnology. These expression vectors were transfected into HEK293Tcells. After transfection, cells expressing G₅-myc-CD40 fusion proteinswere incubated with cells expressing CD40L-SrtA fusion protein,SrtA-PDGFR fusion protein (a not-interacting partner as a negativecontrol), or untransfected cells, for 30 min in presence ofbiotin-LPETGG (SEQ ID NO: 5) peptide. In the case of CD40-CD40L pair,additional controls were investigated, including three mutated versionof CD40L-SrtA (CD40LR₂₀₂E-SrtA, CD40LK₁₄₂E-SrtA, andCD40LK₁₄₂E/R₂₀₂W-SrtA). These mutants are impaired in CD40 binding.

The cells were then washed, stained with Streptavidin and analyzed byflow cytometry. For all the tested ligand-receptor pairs, a stronglabeling of G₅-expressing cells was observed when incubated with thematching interacting partner (e.g., G₅-myc-CD40 and CD40L-SrtA). FIGS.2B, 2C, and 2D. The level of G₅-expressing cell labeling was much higherwhen incubated with cells expressing the wild-type CD40L-SrtA, ascompared to that when incubated with cells expressing one of threemutants. FIGS. 2B, 2C, and 2D. When incubated with the non-interactingmolecule SrtA-PDGFR, only a very limited degree of labeling wasobserved, indicating that ligand-receptor binding/affinity are requiredfor efficient transfer of the labeled SrtA substrate. FIGS. 2B, 2C, and2D.

In the case of CD40-CD40L pair, it was demonstrated by western blotanalysis of the samples that the transfer of the labeled substrateoccurs specifically on the G₅-CD40 molecule (FIG. 2E) as indicated bythe presence in the Streptavidin detection of a defined band at themolecular weight corresponding to G₅-CD40.

Intercellular labeling between engineered CD40-CD40L pair was alsotested in primary murine B and T lymphocytes (FIG. 3A). As observed inHEK293T cells, intercellular labeling of G₅-CD40 expressing B cellsoccurs efficiently upon interaction with CD4⁺ T cells transduced withCD40L-SrtA, but it is present at a very low level whenCD40LK₁₄₂E/R₂₀₂W-SrtA was used. FIGS. 3B and 3C. This result confirmsthat intercellular labeling in the system described herein reflectsligand-receptor interaction and/or affinity.

Example 2: Intercellular Labeling Mediated by Various Ligand-ReceptorPair Interaction

Intercellular labeling mediated by additional ligand-receptor pairinteraction (including CD28/CD80, CTLA4/CD80, CD28/CD86, CTLA4/CD86,PD-1/PD-L1, PD-1/PD-L2, and ICOS/ICOSL) was examined following themethod described in Example 1 above.

Constructs for expressing the ligand/receptor fusion proteins areillustrated in FIG. 4A. These expression vectors were transfected intoHEK293T cells. Cells expressing G₅-containing fusion proteins wereincluded with cells expressing the corresponding interaction partner-Srtfusion proteins min in presence of a biotin-LPETGG (SEQ ID NO: 5)peptide. The cells were then washed, stained with Streptavidin andanalyzed by flow cytometry. As shown in FIGS. 4B and 4C. A stronglabeling of G₅-expressing cells was observed when incubated with cellsexpressing the interacting partner. These results further demonstratethat the cell labelled was mediated by receptor-ligand interaction.

Example 3: Intercellular Labeling in Primary Murine Lymphocytes

Intercellular labeling was also tested in primary murine B and Tlymphocytes overexpressing SrtA/G5 tagged constructs (FIG. 7). Asobserved in HEK293T cells, intercellular labeling of G₅-CD40 expressingB cells occurs efficiently upon interaction with CD4+T OT-II cellsoverexpressing CD40L-SrtA, but it is present at a very low level whenSrtA-PDGFR is used. When OT-II peptide is added to the co-culture,intercellular labeling is efficiently achieved with both CD40L-SrtA andSrtA-PDGFR expressing CD4+ T cells, indicating that cognate interactioncan be tracked also using non interacting molecules (i.e., SrtA-PDFGR).

Example 4: Intercellular Labeling of Ligand-Receptor Interactions InVivo

Interactions between different ligand-receptor pairs expressed byvarious subsets of immune cells are key events in the immune responsebut the tracking of these interactions in the context of a living animalhas never been achieved. To test the application of the method describedherein to follow ligand-receptor interactions in vivo, SrtA-PDGFRtransduced T cells were transferred to a recipient mouse. 24 hours afterthe cell transfer, the mouse was also injected with biotin-LPETGG (SEQID NO: 5) peptide subcutaneously. Inguinal and popliteal lymph nodeswere harvested 1 hour after substrate injection and analyzed by flowcytometry (FIG. 5).

SrtA transduced T cells shown a clear Streptavidin staining (SrtA formsa covalent intermediate with biotin-LPETGG (SEQ ID NO: 5)), indicatingthat SrtA substrate used readily distributed in the analyzed lymph nodesand that enzymatic activity is maintained in the extracellular milieu ofthe living animal.

G₅-CD40 and G₅-CD86 knock-in transgenic mice were constructed. FIG. 6,left panel. The insertion of transgenes were confirmed by sequencinganalysis. FIG. 6, middle panel. The mice were administered withbiotin-LPETGG (SEQ ID NO: 5) and 3 μM SrtA. Lymphocytes were isolatedfrom the treated mice, stained with streptavidin, and subjected to FACSanalysis. As shown in FIG. 6, right panel, lymphocytes isolated from theG₅-CD40 knock-in mice were labeled, while lymphocytes isolated fromcontrol mice treated with 3 μM SrtA or untreated were not labeled.

Further, two gene-targeted mice shown below were generated to test thelabeling strategy described above to track cell:cell interactions inliving animals:

(i) CD40L-SrtA mouse, carrying SrtA gene downstream the last coding exonof Cd401 gene (FIG. 8); and

(ii) G5-CD40 mouse, carrying the genetic sequence encoding five glycineresidues inserted in the second exon of Cd40 gene. (FIG. 9).

The labeling strategy in lymphocytes harvested from the two generatedG5-CD40 and CD40L-SrtA mice noted above was investigated. B and CD4+Tlymphocytes were harvested from G5-CD40+/+ and CD40L-SrtA+/Y OT-II mice,respectively, and cells were co-cultured for 16 hours in presence orabsence of OVA223-239 peptide. Before flow cytometry analysis, cellswere incubated for 1 additional hour with 100 uM Biotin-LPETGG (SEQ IDNO: 5). In this experiment antigen-dependent labeling of B cells wasobserved. Indeed, in absence of OVA223-239 peptide and so in absence ofpMHC-II:TCR cognate interaction and consequent TCR triggering,CD40L-SrtA in CD4+ T cells was not found to express at the cell surface.In presence of OVA223-239 peptide, CD40L-SrtA was observed to bedelivered at the plasma membrane in an OVA223-239 peptide-dose-dependedmanner. Once at the cell surface, CD40L was free to interact with CD40molecules expressed constitutively by B cells and mediate intercellularlabeling.

Example 5: Application of SrtA/G5 Intercellular Labeling Strategy inAntigen Discovery

The SrtA/G5 intercellular labeling system was explored for itsapplication in identifying cognate antigens for T cell receptors withunknown specificities.

A schematic representation of the experimental set-up for studyingintercellular labeling upon B cell:CD4⁺ T cell interaction ex vivo isprovided in FIG. 10, panel A. Briefly, CD40L-SrtA+/OT-II CD4+ T cellsand G5-CD40+/+B cells were isolated and activated. After removal of theactivation stimuli, the CD4+ T cells and B cells were co-cultured for asuitable period. Biotin-LPETGG (SEQ ID NO: 5) was then added to theco-culture and the cells were subject to FACS analysis to examine biotinlabeling of both the T cells and the B cells. FIG. 10, panel B.

Further, intercellular labeling upon interaction of antigen-presentingcells (APCs) such as dendritic cells (DC) and T cells in vivo wasinvestigated as follows. A schematic representation of the experimentalset-up is provided in FIG. 11, panel A. G5-CD40^(+/+) CD45.1⁺ DCs werepulsed with OVA₃₂₃₋₃₃₉ and injected subcutaneously in the footpad ofC57BL/6 mice (1-2×10⁶ DCs per footpad). 24 hours later, the mice werefurther administered intravenously (i.v.) with 5-10×10⁶ CD40L-SrtA^(+/Y)or ^(−/Y) OT-II CD4⁺ T cells. 15 hours after T cell transfer, the micewere injected subcutaneously (s.c.) with 1 μmol of biotin-LPETGG (SEQ IDNO: 5) every 30 min for a total of 4 hours. Popliteal lymphocytes (LNs)were then harvested and analyzed by flow cytometry. As shown in FIG. 11,panel B, formation of biotin-LPET:SrtA covalent intermediate inCD40L-Srt^(+/Y) OT-II CD4⁺ T cells was observed (left panel) andlabeling was specifically detected in antigen-pulsed DCs (right panel).

The experiments presented above show how SrtA/G5 engineered surfacemolecules can be used to track intercellular interactions in vivo inmice. Using engineered G5-CD40 and CD40L-SrtA molecules, labeling of Bcells and DCs by CD4⁺ T cells upon cognate interaction, i.e., uponspecific pMHC-II:TCR recognition, were achieved. Accordingly, thismethod can be used to discover cognate antigen recognized by TCRs whosespecificity is unknown.

An exemplary system composed of the following components may be used toachieve this aim:

1. A collection of cell lines (human, murine or from any other speciesof interest) derived from antigen presenting cells (APCs; B cells, DCs,macrophages) engineered to express:

-   -   MHC-I and MHC-II alleles (human, murine or from any other        species of interest);    -   a protein at the plasma membrane carrying N-terminal glycine        residue(s); and    -   a library of cDNAs (human, murine or from any other species of        interest).

2. A T cell line engineered to express:

-   -   a TCR whose specificity need to be determined    -   a membrane protein carrying in its extracellular portion SrtA        enzyme

3. A substrate for SrtA enzyme containing the amino acid sequence LPXTG(SEQ ID NO: 1) conjugated with biotin, a fluorophore or other detectablelabel.

Different SrtA/G5 pairs could be used to achieve labeling of APCs uponpMHC:TCR recognition. One strategy is to functionalize with SrtA/G5ligand-receptor pairs which are upregulated upon pMHC:TCR recognition,as in the case of CD40-CD40L. Alternatively, labeling upon pMHC:TCRrecognition can be achieved using a membrane bound form of SrtA (e.g.,SrtA fused to PDGFR transmembrane domain) in combination with a G5tagged protein that is upregulated upon cognate interaction (i.e., CD40,CD80, CD86).

In the assay, engineered APCs and T cells can be co-cultured in vitro inpresence of a labeled SrtA substrate. Upon cognate interaction (specificpMHC:TCR recognition) between APCs and T cells intercellular labelingoccurs and a subpopulation of APCs are labeled. These cells can berecovered by FACS sorting. If necessary, it is possible to enrich thepopulation of labeled APCs before sorting using commercially availableimmuno-magnetic isolation, using specific reagents recognizing biotin orfluorophores. After sorting, exogenous DNA sequence can be identifiedby, e.g., PCR amplification and sequencing, revealing in this way theantigen specificity of the TCR of interest.

Antigens identified by the methods described herein as recognizable by Tcells of interest, for example, T cell isolated from a tumor site orinfectious site, may be useful as vaccine antigens for treating thecorresponding cancer or infection.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps.

Where ranges are given, endpoints are included. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or sub-range withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

What is claimed is:
 1. An intercellular labeling method comprising: (i)providing a first cell expressing a first polypeptide on the surface ofthe first cell, the first polypeptide comprising a sortase acceptorpeptide, which is located at the N-terminus of the first polypeptide;(ii) providing a second cell expressing a second polypeptide on thesurface of the second cell, the second polypeptide comprising a sortaseor an active fragment thereof; and (iii) contacting the first cell withthe second cell in the presence of a sortase substrate comprising asortase recognition sequence, wherein the sortase substrate isassociated with a detectable label; wherein upon interaction between thefirst cell and the second cell, the sortase or the active fragmentthereof links the sortase substrate to the first polypeptide, therebylabeling the first cell expressing the first polypeptide.
 2. Theintercellular labeling method of claim 1, wherein the first polypeptideis a fusion polypeptide comprising the sortase acceptor peptide and onemember of a receptor-ligand pair; and wherein the second polypeptide isa fusion polypeptide comprising the sortase or the active fragmentthereof and the other member of the receptor-ligand pair.
 3. Theintercellular labeling method of claim 1, wherein the first cell, thesecond cell, or both are immune cells.
 4. The intercellular labelingmethod of claim 3, wherein the first cell, the second cell, or both areT cells, B cells, dendritic cells, macrophages, or natural killer cells.5. The intercellular labeling method of claim 4, wherein the first cellis a T cell, and the second cell is a B cell, or vice versa.
 6. Theintercellular labeling method of claim 2, wherein the receptor-ligandpair is selected from the group consisting of: CD40 and CD40L, CD80 andCD28, CD80 and CTLA4, CD86 and CD28, CD86 and CTLA4 PD-1 and PD-L1, PD-1and PD-L2, and ICOS and ICOSL.
 7. The intercellular labeling method ofclaim 1, wherein the detectable label is biotin or a fluorescent dye. 8.The intercellular labeling method of claim 1, wherein the sortase is asortase A.
 9. The intercellular labeling method of claim 8, wherein thesortase is a mutant sortase A that exhibits improved catalytic activityas compared to the wild-type counterpart.
 10. The intercellular labelingmethod of claim 9, wherein the mutant comprises one or more mutations ofP94R or P94S, S102C, A104H, E105D, K138P, K152I, D160K or D160N, K162H,T164N, D165A, K173E, I182V, K190E, and K196S or K196T.
 11. Theintercellular labeling method of claim 10, wherein the mutant sortase Acontains mutations P94S, D160N, and K196T.
 12. The intercellularlabeling method of claim 1, wherein the sortase recognition sequence isLPXTG (SEQ ID NO: 1), in which X is any amino acid residue.
 13. Theintercellular labeling method of claim 12, wherein the sortaserecognition sequence is LPETG (SEQ ID NO: 2).
 14. The intercellularlabeling method of claim 1, wherein the sortase acceptor peptide is anoligoglycine.
 15. The intercellular labeling method of claim 14, whereinthe oligoglycine consists of 1-5 glycine residues.
 16. The intercellularlabeling method of claim 2, wherein the first polypeptide comprises CD40and the N-terminal sortase acceptor peptide, which is oligoglycine GGGGG(SEQ ID NO: 3), and the second polypeptide comprises CD40L, which isfused to the sortase or the active fragment thereof.
 17. Theintercellular labeling method of claim 16, wherein the first cell is a Bcell, and the second cell is a T cell.
 18. The intercellular labelingmethod of claim 1, wherein the first polypeptide, the secondpolypeptide, or both further comprise a protein tag.
 19. Theintercellular labeling method of claim 1, wherein the method isperformed in vitro.
 20. The intercellular labeling method of claim 1,wherein the method is performed in vivo.
 21. The intercellular labelingmethod of claim 20, wherein the first cell, the second cell, or both areendogenous cells of a transgenic animal.
 22. The intercellular labelingmethod of claim 21, wherein the first cell is an endogenous cell of atransgenic animal, and the second cell is constructed in vitro andtransferred into the same transgenic animal, or vice versa.
 23. Theintercellular labeling method of claim 21, wherein transgenic animal isa transgenic mouse, rat, or rabbit.
 24. The intercellular labelingmethod of claim 20, wherein the first cell, the second cell, or both areconstructed in vitro and transferred into a subject.
 25. Theintercellular labeling method of claim 24, wherein the subject is amouse, a rabbit, a rat, or a monkey.
 26. The intercellular labelingmethod of claim 21, wherein the peptide comprising the sortaserecognition sequence is administered to the transgenic animal or thesubject.
 27. The intercellular labeling method of claim 20, wherein thecontacting step is carried out in a germinal center.
 28. Theintercellular labeling method of claim 2, wherein the contacting step isperformed in the presence of a candidate compound, and the methodfurther comprises assessing whether the candidate compound modulates theinteraction between the two members of the receptor-ligand pair, whereina change of the labeling of the first cell in the presence of thecandidate compound indicates that the compound is a modulator of thereceptor-ligand pair.
 29. The intercellular labeling method of claim 1,wherein the first cell is an antigen-presenting cell (APC) thatexpresses a MHC class I molecule, a MHC class II molecule, or both; andthe second cell is a T cell that expresses a T cell receptor (TCR)molecule.
 30. The intercellular labeling method of claim 29, wherein theAPC cell is a B cell, a dendritic cell, a macrophage, or a B cell. 31.The intercellular labeling method of claim 29, wherein the APC isengineered to further express a polypeptide encoded by a member of acDNA library.
 32. The intercellular labeling method of claim 29, whereinstep (i) is performed by providing a plurality of APCs whichcollectively express polypeptides encoded by the cDNA library; andwherein step (iii) is performed by contacting the plurality of the APCswith the T cell in the presence of the sortase substrate.
 33. Theintercellular labeling method of claim 32, further comprising isolatingthe labeled APCs produced in step (iii).
 34. The intercellular labelingmethod of claim 33, further comprising identifying the member of thecDNA library that is expressed in the labeled APCs for determiningantigen specificity of the TCR expressed on the T cell.
 35. A kit forintercellular labeling, comprising: (i) a first cell expressing a firstpolypeptide on its surface, the first polypeptide comprising a sortaseacceptor peptide, which is located at the N-terminus of the firstpolypeptide; and (ii) a second cell expressing a second polypeptide onits surface, the second polypeptide comprising a sortase or an activefragment thereof.
 36. The kit of claim 35, wherein the first polypeptideis a fusion polypeptide comprising the sortase acceptor peptide and onemember of a receptor-ligand pair; and wherein the second polypeptide isa fusion polypeptide comprising the sortase or the active fragmentthereof and the other member of the receptor-ligand pair.
 37. The kit ofclaim 35, wherein the first cell, the second cell, or both are immunecells.
 38. The kit of claim 37, wherein the first cell, the second cell,or both are T cells, B cells, dendritic cells, macrophages, or naturalkiller cells.
 39. The kit of claim 38, wherein the first cell is a Tcell and the second cell is a B cell, or vice versa.
 40. The kit ofclaim 36, wherein the receptor-ligand pair is selected from the groupconsisting of: CD40 and CD40L, CD80 and CD28, CD80 and CTLA4, CD86 andCD28, CD86 and CTLA4, PD-1 and PD-L1, PD-1 and PD-L2, and ICOS andICOSL.
 41. The kit of claim 35, wherein the sortase is a sortase A. 42.The kit of claim 35, wherein the sortase is a mutant sortase A thatexhibits improved catalytic activity as compared to the wild-typecounterpart.
 43. The kit of claim 42, wherein the mutant sortase Acomprises one or more mutations of P94R or P94S, S102C, A104H, E105D,K138P, K152I, D160K or D160N, K162H, T164N, D165A, K173E, I182V, K190E,and K196S or K196T.
 44. The kit of claim 43, wherein the mutant sortaseA contains mutations P94S, D160N, and K196T.
 45. The kit of claim 35,wherein the sortase acceptor peptide is an oligoglycine.
 46. The kit ofclaim 45, wherein the oligoglycine consists of 1-5 glycine residues. 47.The kit of claim 46, wherein the first polypeptide comprises CD40 andthe N-terminal sortase acceptor, which is oligoglycine GGGGG (SEQ ID NO:3), and the second polypeptide comprises CD40L, which is fused to thesortase at the C-terminus.
 48. The kit of claim 47, wherein the firstcell is a B cell, and the second cell is a T cell, or vice versa. 49.The kit of claim 35, wherein the first polypeptide, the secondpolypeptide, or both further comprise a protein tag.
 50. The kit ofclaim 35, wherein the kit further comprises a sortase substratecomprising a sortase recognition sequence, wherein the sortase substrateis associated with a detectable label.
 51. The kit of claim 50, whereinthe sortase recognition sequence is LPXTG (SEQ ID NO: 1), in which X isany amino acid residue.
 52. The kit of claim 51, wherein the sortaserecognition sequence is LPETG (SEQ ID NO: 2).
 53. The kit of claim 35,wherein the first cell is an antigen-presenting cell (APC) thatexpresses a MHC class I molecule, a MHC class II molecule, or both; andthe second cell is a T cell that expresses a T cell receptor (TCR)molecule.
 54. The kit of claim 53, wherein the APC cell is a B cell, adendritic cell, a macrophage, or a B cell.
 55. The kit of claim 53,wherein the APC is engineered to further express a polypeptide encodedby a member of a cDNA library.
 56. The kit of claim 55, wherein (i)contains a plurality of APCs which collectively express polypeptidesencoded by the cDNA library.
 57. The kit of claim 53, further comprisinga sortase substrate comprising a sortase recognition sequence, whereinthe sortase substrate is associated with a detectable label.
 58. The kitof claim 57, wherein the sortase recognition sequence is LPXTG (SEQ IDNO: 1), in which X is any amino acid residue.
 59. The kit of claim 58,wherein the sortase recognition sequence is LPETG (SEQ ID NO: 2).
 60. Anon-human animal, comprising: (i) a first cell expressing a firstpolypeptide on its surface, the first polypeptide comprising a sortaseacceptor peptide, which is located at the N-terminus of the firstpolypeptide; (ii) a second cell expressing a second polypeptide on itssurface, the second polypeptide comprising a sortase, or (iii) both (i)and (ii).
 61. The non-human animal of claim 60, wherein the firstpolypeptide is a fusion polypeptide comprising the sortase acceptorpeptide and one member of a receptor-ligand pair; and wherein the secondpolypeptide is a fusion polypeptide comprising the sortase or the activefragment thereof and the other member of the receptor-ligand pair. 62.The non-human animal of claim 60, wherein the animal is a transgenicanimal, in which a gene encoding the first polypeptide, a gene encodingthe second polypeptide, or both are inserted into the genome of theanimal.
 63. The non-human animal of claim 60, wherein the animal is atransgenic animal, in which a nucleic acid sequence encoding the sortaseacceptor peptide is inserted into the endogenous locus encoding the onemember of the ligand-receptor pair for expression of the firstpolypeptide.
 64. The animal of claim 60, wherein the animal is atransgenic animal, in which a nucleic acid sequence encoding the sortaseis inserted into the endogenous locus encoding the other member of theligand-receptor pair for expression of the second polypeptide.
 65. Thenon-human animal of claim 60, wherein the animal is a transgenic animal,in which the gene encoding the first polypeptide is inserted into thegenome of the animal, and the second cell that expresses the secondpolypeptide is constructed in vitro and transferred into the animal. 66.The non-human animal of claim 60, wherein the animal is a transgenicanimal, in which the gene encoding the second polypeptide is insertedinto the genome of the animal and the first cell that expresses thefirst polypeptide is constructed in vitro and transferred into theanimal.
 67. The non-human animal of claim 60, wherein the transgenicanimal is a transgenic mouse, rat, or rabbit.
 68. The non-human animalof claim 60, wherein both the first cell and the second cell areconstructed in vitro and transferred into the animal.
 69. The non-humananimal of claim 60, wherein the first cell, the second cell, or both areimmune cells.
 70. The non-human animal of claim 69, wherein the firstcell, the second cell, or both are T cells, B cells, dendritic cells,macrophages, or natural killer cells.
 71. The non-human animal of claim70, wherein the first cell is a T cell, and the second cell is a B cell,or vice versa.
 72. The non-human animal of claim 61, wherein thereceptor-ligand pair is selected from the group consisting of: CD40 andCD40L, CD80 and CD28, CD80 and CTLA4, CD86 and CD28, CD86 and CTLA4,PD-1 and PD-L1, PD-1 and PD-L2, and ICOS and ICOSL.
 73. The non-humananimal of claim 60, wherein the sortase is a sortase A.
 74. Thenon-human animal of claim 60, wherein the sortase is a mutant sortase Athat exhibits improved catalytic activity as compared to the wild-typecounterpart.
 75. The non-human animal of claim 74, wherein the mutantsortase A comprises one or more mutations of P94R or P94S, S102C, A104H,E105D, K138P, K152I, D160K or D160N, K162H, T164N, D165A, K173E, I182V,K190E, and K196S or K196T.
 76. The non-human animal of claim 75, whereinthe mutant sortase A contains mutations P94S, D160N, and K196T.
 77. Thenon-human animal of claim 60, wherein the sortase acceptor peptide is anoligoglycine.
 78. The non-human animal of claim 77, wherein theoligoglycine consists of 1-5 glycine residues.
 79. The non-human animalof claim 60, wherein the first polypeptide comprises CD40 and theN-terminal acceptor peptide, which is GGGGG (SEQ ID NO: 3), and thesecond polypeptide comprises CD40L, which is fused to the sortase at theC-terminus.
 80. The non-human animal of claim 79, wherein the first cellis a B cell and the second cell is a T cell.
 81. The non-human animal ofclaim 60, wherein the non-human animal is a transgenic non-human mammalthat comprises one or more human immunoglobulin genes or a portionthereof.
 82. The non-human animal of claim 81, wherein the non-humanmammal is a transgenic mouse or transgenic rat.
 83. The non-human animalof claim 60, wherein the non-human mammal comprises a humanized immunesystem.
 84. The non-human animal of claim 60, wherein the non-humananimal is a transgenic mouse expressing a fusion polypeptide comprisingsortase A and CD40L.
 85. The non-human animal of claim 60, wherein thenon-human animal is a transgenic mouse expressing a fusion polypeptidecomprising GGGGG (SEQ ID NO: 3) and CD40, wherein the GGGGG (SEQ ID NO:3) fragment is located at the N-terminus of the fusion polypeptide. 86.A nucleic acid comprising a nucleotide sequence that encodes apolypeptide comprising a sortase and a member of a ligand-receptor pair.87. The nucleic acid of claim 86, wherein the sortase is a sortase A.88. The nucleic acid of claim 87, wherein the sortase A is a mutant of awild-type sortase A, which has improved catalytic activity as comparedto the wild-type counterpart.
 89. The nucleic acid of claim 88, whereinthe mutant comprises one or more mutations of P94R or P94S, S102C,A104H, E105D, K138P, K152I, D160K or D160N, K162H, T164N, D165A, K173E,I182V, K190E, and K196S or K196T.
 90. The nucleic acid of claim 89,wherein the mutant contains mutations of P94S, D160N, and K196T.
 91. Thenucleic acid of claim 86, wherein the polypeptide further comprises aprotein tag.
 92. A vector comprising the nucleic acid of claim
 86. 93.The vector of claim 92, wherein the vector is an expression vector. 94.A host cell comprising the vector of claim
 92. 95. A method foridentifying a B cell expressing a high affinity B cell receptor (BCR) toan antigen, the method comprising: (i) providing a mammal that comprises(a) a plurality of B cells expressing a first polypeptide on itssurface, the first polypeptide comprising a sortase acceptor peptide,which is located at the N-terminus of the first polypeptide, and (b) aplurality of T cells each expressing a second polypeptide on itssurface, the second polypeptide comprising a sortase or an activefragment thereof; (ii) administering to the mammal an effective amountof a sortase substrate comprising a sortase recognition sequence,wherein the sortase substrate is associated with a detectable label;(iii) isolating lymphocytes from a germinal center of the animal; and(iv) identifying a B cell that is conjugated to the detectable label,wherein the B cell thus identified expresses a high affinity BCR to anantigen.
 96. The method of claim 95, wherein the first polypeptide is afusion polypeptide comprising the sortase acceptor peptide and onemember of a receptor-ligand pair, the member being a B cell surfaceprotein; and wherein the second polypeptide is a fusion polypeptidecomprising the sortase or the active fragment thereof and the othermember of the receptor-ligand pair, the other member being a T cellsurface receptor.
 97. The method of claim 95, wherein the mammal is atransgenic mammal, in which a gene encoding the first polypeptide, agene encoding the second polypeptide, or both are inserted into thegenome of the mammal.
 98. The method of claim 95, wherein the mammal isa transgenic animal, in which a nucleic acid sequence encoding thesortase acceptor peptide is inserted into the endogenous locus encodingthe one member of the ligand-receptor pair for expression of the firstpolypeptide.
 99. The method of claim 95, wherein the mammal is atransgenic animal, in which a nucleic acid sequence encoding the sortaseis inserted into the endogenous locus encoding the other member of theligand-receptor pair for expression of the second polypeptide.
 100. Themethod of claim 95, wherein the mammal is a transgenic mammal, in whichthe gene encoding the first polypeptide and the gene encoding the secondpolypeptide are both inserted into the genome of the mammal.
 101. Themethod of claim 95, wherein the mammal is a transgenic mammal, in whichthe gene encoding the first polypeptide is inserted into the genome ofthe mammal and expressed on naïve B cells and the plurality of T cellsthat expresses the second polypeptide are constructed in vitro andtransferred into the mammal.
 102. The method of claim 95, wherein thetransgenic mammal is a transgenic mouse, rat, or rabbit.
 103. The methodof claim 95, wherein the sortase is a sortase A.
 104. The method ofclaim 95, wherein the sortase is a mutant sortase A that exhibitsimproved catalytic activity as compared to the wild-type counterpart.105. The method of claim 104, wherein the mutant of sortase A comprisesone or more mutations of P94R or P94S, S102C, A104H, E105D, K138P,K152I, D160K or D160N, K162H, T164N, D165A, K173E, I182V, K190E, andK196S or K196T.
 106. The method of claim 105, wherein the mutant sortaseA contains mutations P94S, D160N, and K196T.
 107. The method of claim95, wherein the sortase recognition sequence is LPXTG (SEQ ID NO: 1), inwhich X is any amino acid residue.
 108. The method of claim 107, whereinthe sortase recognition sequence is LPETG (SEQ ID NO: 2).
 109. Themethod of claim 95, wherein the sortase acceptor peptide is anoligoglycine.
 110. The method of claim 109, wherein the oligoglycineconsists of 1-5 glycine residues.
 111. The method of claim 95, whereinthe mammal is immunized with an antigen of interest.
 112. The method ofclaim 95, the method further comprising isolating one or more nucleicacid encoding at least a portion of a heavy chain variable region, atleast a portion of a light chain variable region, or both of the BCRfrom the B cell that is conjugated to the detectable label.
 113. Themethod of claim 112, wherein the at least a portion of the heavy chainvariable region, the at least a portion of the light chain variableregion, or both encode at least one complementarity determining regionof the BCR.
 114. The method of claim 113, further comprising sequencingthe at least a portion of the heavy chain variable region, the at leasta portion of the light chain variable region, or both.
 115. The methodof claim 95, the method further comprising producing a hybridoma cellderived from the B cell that is conjugated to the detectable label,wherein the hybridoma cell produces high affinity antibodies to theantigen.
 116. The method of claim 115, further comprising culturing thehybridoma cell for producing the antibodies.
 117. The method of claim95, wherein the non-human mammal is a transgenic non-human mammal thatcomprise one or more human immunoglobulin genes, or a portion thereof.118. The method of claim 117, wherein the non-human mammal is atransgenic mouse or transgenic rat.
 119. The method of claim 95, whereinthe non-human mammal comprises a humanized immune system.
 120. A methodfor identifying a binding partner of a protein of interest, the methodcomprising: (i) providing a first population of cells expressing aplurality of polypeptides, each of which expresses a sortase acceptorpeptide and a candidate protein; (ii) providing a second population ofcells expressing a sortase or an active fragment thereof, and theprotein of interest; (iii) contacting the first population of cells withthe second population of cells in the presence of a sortase substratecomprising a sortase recognition sequence, wherein the peptide isassociated with a detectable label; (iv) detecting labeling of cells inthe first population of cells; and (iv) identifying a binding partner ofthe protein of interest, wherein a candidate protein is a bindingpartner of the protein of interest, if the cell that expresses apolypeptide comprising the candidate protein is labeled in step (iii).121. The method of claim 120, wherein in (i), the sortase acceptorpeptide and the candidate protein are covalently linked to form a fusionpolypeptide.
 122. The method of claim 120, wherein in (ii), the sortaseor the active fragment thereof and the protein of interest arecovalently linked to form a fusion polypeptide.
 123. The method of claim122, wherein the protein of interest is a receptor of an immune cell.124. The method of claim 120, wherein the immune cell is a T cell, a Bcell, a dendritic cell, a macrophage, or a natural killer cell.
 125. Themethod of claim 120, wherein the detectable label is biotin or afluorescent dye.
 126. The method of claim 120, wherein the sortase is asortase A.
 127. The method of claim 120, wherein the sortase is a mutantsortase A that exhibits improved catalytic activity as compared to thewild-type counterpart.
 128. The method of claim 127, wherein the mutantsortase A comprises one or more mutations of P94R or P94S, S102C, A104H,E105D, K138P, K152I, D160K or D160N, K162H, T164N, D165A, K173E, I182V,K190E, and K196S or K196T.
 129. The method of claim 128, wherein themutant sortase A contains mutations P94S, D160N, and K196T.
 130. Themethod of claim 120, wherein the sortase recognition sequence is LPXTG(SEQ ID NO: 1), in which X is any amino acid residue.
 131. The method ofclaim 130, wherein the sortase recognition sequence is LPETG (SEQ ID NO:2).
 132. The method of claim 120, wherein the sortase acceptor peptideis an oligoglycine.
 133. The method of claim 132, wherein theoligoglycine consists of 1-5 glycine residues.
 134. The method of claim120, wherein the polypeptide comprising the protein of interest, thepolypeptides comprising the candidate proteins, or both further comprisea protein tag.
 135. The method of claim 120, wherein the method isperformed in vitro.